Mitochondrial markers of neurodegenerative diseases

ABSTRACT

Disclosed is an in vitro method to diagnose or determine the risk of developing a neurodegenerative disease in a subject based on the determination of the methylation pattern in certain regions of mitochondrial DNA from the subject or the determination of the nucleotide at the polymorphic position 16519 in the mitochondrial DNA of the subject. Also, disclosed are nucleic acids suitable for the in vitro method to diagnose or determine the risk of developing a neurodegenerative disease in a subject.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

This application is the U.S. National Phase of International ApplicationNo. PCT/ES2015/070230, filed Mar. 27, 2015, designating the U.S. andclaiming priority to Spain Application No. P201430444, filed Mar. 28,2014. Any and all applications for which a foreign or domestic priorityclaim is identified here or in the Application Data Sheet as filed withthe present application are hereby incorporated by reference under 37CFR 1.57.

FIELD OF THE INVENTION

The present invention is classified as part of the diagnosis methods ofneurological diseases.

BACKGROUND OF THE INVENTION

A considerable amount of neurodegenerative diseases are caused by orassociated with altered mitochondrial function.

Alzheimer's disease (AD) and Parkinson's disease (PD) are within thisgroup. The pathophysiological characteristics of AD and PD are relatedto deposits of aggregated protein. Specifically, AD is associated withthe formation of intracellular phosphorylated tau aggregates inneurofibrillary tangles and extracellular aggregates of β-amyloidpeptide in the senile plaques and PD is associated with the formation ofabnormal α-synuclein aggregates that constitute the main component ofthe so called Lewy bodies and Lewy neurites.

It is accepted that Alzheimer's patients show decreased levels of thesubunit ND4 in their brain tissue and that Parkinson's patients showreduced levels of ND6 in the substantia nigra. Furthermore, geneticstudies have identified mutations in several COX genes and in the D-loopregion as well as deletions of mtDNA in the brains of subjects with ADand in the substantial nigra of subjects with PD.

Different methods and strategies have been developed in the technologyfor diagnosis, prediction of onset and the development ofneurodegenerative diseases, and specifically of AD and PD. In that way,diagnosis methods have been described for neurodegenerative diseasesbased on the identification of mutations in mitochondrial DNA throughthe employment of the RFLP (restriction fragment length polymorphism)technique or of other related techniques. The WO98038334 documentdescribes a method of AD diagnosis based on the identification ofmutations in COX genes. It has also proposed a method of PD diagnosis ina subject through the identification of single nucleotide polymorphismsin mitochondrial DNA samples of a subject (WO 2000063441). Otherdocuments of the prior art describe diagnosis methods of Alzheimer's orParkinson's disease based on the identification of polymorphisms innuclear protein encoding genes that control the process of mitochondrialtranscription. Despite the efforts that have been made to date, thereremains a need for reliable methods of diagnosis for neurodegenerativediseases such as AD and PD, as well for the diagnosis of the stage ofsuch diseases and to prognosticate the evolution of the same.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to an in vitro method todiagnose or to determine the risk of development of a neurodegenerativedisease selected from Alzheimer's disease and Parkinson's disease in asubject that contains, in a sample from said subject containingmitochondrial DNA, the methylation pattern in the D-loop region and/orin the ND1 gene, wherein the methylation pattern is determined in atleast one site selected from the group formed by:

-   -   i. the CpG sites in the D-loop region shown in Table 1    -   ii. the CpG sites in the ND1 gene shown in Table 2,    -   iii. the CHG sites in the D-loop region shown in Table 3,    -   iv. the CHG sites in the ND1 gene shown in Table 4, and/or    -   v. the CHH sites in the D-loop region shown in Table 5,        wherein a hypermethylation in at least one of said CpG sites in        the D-loop region, a hypermethylation in at least one of said        CHG sites in the D-loop region, a hypermethylation in at least        one of said CHH sites in the D-loop region, a hypomethylation in        at least one of said CpG sites in the ND1 gene and/or a        hypomethylation in at least one of said CHG sites in the ND1        gene it is indicative that the subject suffers from Alzheimer's        disease or that the subject has an elevated risk of developing        Alzheimer's disease or        wherein a hypomethylation in at least one of said CpG sites in        the D-loop region, a hypomethylation in at least one of said CHG        sites in the D-loop region and/or a hypomethylation in at least        one of said CHH sites in the D-loop region it is indicative that        the subject suffers from Parkinson's disease or that the subject        has an elevated risk of developing Parkinson's disease.

In a second aspect, the invention relates to an in vitro method toselect a subject for submission to preventive treatment of aneurodegenerative disease selected from Alzheimer's disease andParkinson's disease in a subject that contains, in a sample from saidsubject containing mitochondrial DNA, the methylation pattern in theD-loop region and/or in the ND1 gene, where the methylation pattern isdetermined in at least one site selected from the group formed by:

-   -   i. the CpG sites in the D-loop region shown in Table 1,    -   ii. the CpG sites in the ND1 gene shown in Table 2;    -   iii. the CHG sites in the D-loop region shown in Table 3,    -   iv. the CHG sites in the ND1 gene shown in Table 4, and/or    -   v. the CHH sites in the D-loop region shown in Table 5        wherein a hypermethylation in at least one of said CpG sites in        the D-loop region, a hypermethylation in at least one of said        CHG sites in the D-loop region, a hypermethylation in at least        one of said CHH sites in the D-loop region, a hypomethylation in        at least one of said CpG sites in the ND1 gene and/or a        hypomethylation in at least one of said CHG sites in the ND1        gene is indicative that the subject is eligible to receive        treatment aimed at preventing Alzheimer's disease or        wherein a hypomethylation in at least one of said CpG sites in        the D-loop region, a hypomethylation in at least one of said CHG        sites of the D-loop region and/or a hypomethylation in at least        one of said CHH sites in the D-loop region is indicative that        the subject is eligible to receive treatment aimed at preventing        Parkinson's disease.

In a third aspect, the invention relates to an in vitro method tomonitor the progression of a neurodegenerative disease selected fromAlzheimer's disease or Parkinson's disease in a subject, comprising:

-   -   a) determining in a sample from said subject containing        mitochondrial DNA the methylation pattern in the D-loop region,        and/or in the ND1 gene, wherein the methylation pattern is        determined in at least one site selected from the group formed        by:        -   i. the CpG sites in the D-loop region shown in Table 1,        -   ii. the CpG sites in the ND1 gene shown in Table 2;        -   iii. the CHG sites in the D-loop region shown in Table 3,        -   iv. the CHG sites in the ND1 gene shown in Table 4, and/or        -   v. the CHH sites in the D-loop region shown in Table 5    -   b) comparing the methylation pattern determined in step a) with        said methylation pattern obtained in an earlier stage of the        disease,    -   wherein a hypomethylation in at least one of said CpG sites in        the D-loop region, a hypomethylation in at least one of the said        CHG sites in the D-loop region, a hypomethylation in at least        one of said CHH sites in the D-loop region, a hypomethylation in        at least one of said CpG sites in the ND1 gene and/or a        hypomethylation in at least one of said CHG sites in the ND1        gene with respect to said methylation pattern determined in an        earlier stage of disease is indicative of the advance of        Alzheimer's disease; and    -   wherein a hypomethylation in at least one of said CpG sites in        the D-loop region, a hypomethylation in at least one of said CHG        sites in the D-loop region and/or a hypomethylation in at least        one of the said CHH sites in the D-loop region with respect to        said determined methylation pattern at an earlier stage of the        disease, is indicative of the advance of Parkinson's disease.

In a fourth aspect, the invention relates to an in vitro method todiagnose or determine the risk of development of Alzheimer's disease ina subject that contains in a sample comprising mitochondrial DNA fromsaid subject, the nucleotide at the polymorphic position 16519 accordingto the sequence defined under the accession number NC_012920 in the NCBIdatabase, wherein the detection of nucleotide C at said polymorphicposition or presence of the nucleotide C at said polymorphic position inat least 60% of the molecules of mitochondrial DNA of the subject isindicative that the subject suffers from said disease or that thesubject has an elevated risk of developing the disease.

In a fifth aspect, the invention relates to an in vitro method to selecta subject to be submitted to preventive treatment for Alzheimer'sdisease, that involves determining in a sample containing mitochondrialDNA from said subject, the nucleotide at the polymorphic position 16519according to the sequence defined under accession number NC_012920 inthe NCBI database, where the detection of nucleotide C at saidpolymorphic position or the presence of the nucleotide C at saidpolymorphic position in at least 60% of the mitochondrial DNA moleculesfrom said subject is indicative that the subject is eligible to receivetreatment aimed at preventing Alzheimer's disease.

In a sixth aspect, the invention relates to a nucleic acid selected fromthe group formed by:

-   -   (i) a nucleic acid comprising at least 9 contiguous nucleotides        in a region of Mitochondrial DNA where said region comprises at        least one methylation site selected from the group formed by:        -   a) the CpG sites in the D-loop region shown in Table 1,        -   b) the CpG sites in the ND1 gene shown in Table 2;        -   c) the CHG sites in the D-loop region shown in Table 3,        -   d) the CHG sites in the ND1 gene shown in Table 4, and        -   e) the CHH sites in the D-loop region shown in table 5,    -   (ii) a nucleic acid comprising at least 9 contiguous nucleotides        in a region of mitochondrial DNA region where said region        comprises at least one methylation site selected from the group        formed by:        -   a) the CpG sites in the D-loop region shown in Table 1,        -   b) the CpG sites in the ND1 gene shown in Table 2;        -   c) the CHG sites in the D-loop region shown in Table 3,        -   d) the CHG sites in the ND1 gene shown in Table 4, and        -   e) the CHH sites in the D-loop region shown in Table 5.            where the position corresponding to the cytosine in the CpG,            CHG or CHH site is uracil; and    -   (iii) a polynucleotide that hybridizes specifically with the        nucleic acids of (i) or (ii)

In a seventh aspect, the invention relates to a kit comprising at leastone oligonucleotide capable of specifically hybridizing and in amethylation-dependent manner with a mitochondrial DNA sequencecomprising a methylation site selected from the group formed by:

-   -   i. the CpG sites in the D-loop region shown in Table 1,    -   ii. the CpG sites in the ND1 gene shown in Table 2;    -   iii. the CHG sites in the D-loop region shown in Table 3,    -   iv. the CHG sites in the ND1 gene shown in Table 4, and/or    -   v. the CHH sites in the D-loop region shown in Table 5

In an eighth aspect, the invention relates to a kit comprising at leastone oligonucleotide capable of specifically hybridizing at position 5′or at position 3′ with respect to a methylation site in the selectedmitochondrial DNA from the group formed by:

-   -   (i) the CpG sites in the D-loop region shown in Table 1,    -   (ii) the CpG sites in the ND1 gene shown in Table 2;    -   (iii) the CHG sites in the D-loop region shown in Table 3,    -   (iv) the CHG sites in the ND1 gene shown in Table 4, and/or    -   (v) the CHH sites in the D-loop region shown in Table 5        wherein the methylated cytosine in said position has converted        to uracil or to another base that is distinguishable from        cytosine in its hybridization properties.

Finally, in a ninth aspect, the invention refers to the use of the kitsdefined in the seventh and eight inventive aspects to determine themethylation pattern of mitochondrial DNA and to determine the diagnosisneurodegenerative disease in a subject selected from Alzheimer's diseaseand Parkinson's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Graphics Log 2 (OR) for CpG (A) CHG (B) and CHH (C) sites in theamplicon D-Loop in the entorhinal cortex in AD pathology related cases.Methylation sites of 5′ to 3′ are represented in the x-axis. The sitesmarked with a diamond are differentially methylated sites (FDR<0.05).Dots are OR estimate values, one for each site, and the band is the bandof union of all the confidence intervals of 95%. C: samples control, AD:Alzheimer's Disease.

FIG. 2: Grafics Log 2(OR) for CpG (A) CHG (B) and CHH (C) sites in theND1 amplicon in the entorhinal cortex of AD pathology related cases. InMethylation sites from 5′ to 3′ are represented in the x-axis. Theplaces marked with a diamond are differentially methylated sites ofunion of all the confidence intervals of 95%. C: control samples, AD:Alzheimer's disease.

FIG. 3: Graphics Log 2 (OR) for CpG and non-CpG (CHG and CHH) sites inthe amplicon D-loop in the substantia nigra in patients with PD. In thex-axis are Methylation sites from 5′ to 3′ are represented in thex-axis. The sites marked with a diamond are differentially methylatedsites (FDR<0.05). Dots are estimated OR values, one for each site, andthe band is the band of union of all the confidence intervals of 95%. C:control samples, PD: Parkinson's disease.

FIG. 4: Graphics log 2 (OR) for CpG (A) and CHG (B) sites in theamplicon D-Loop in the frontal cortex of APP/PS1 mice and wild mice (WT)of three, six and twelve months. methylation sites sorted by 5′ to 3′are represented in the x-axis. The sites marked with a diamond aredifferentially methylated sites (FDR<0.05). Dots are estimated ORvalues, one for each site, and the band is the band of union of all theconfidence intervals of 95%. C: control samples, WT: wild, TG:Transgenic.

FIG. 5: Graphics Log 2 (OR) for CG (A) CHG (B) and CHH (C) sites in theamplicon D-Loop in the frontal cortex of APP/PS1 of mice three, six andtwelve months. Methylation sites sorted by 5′ to 3′ are represented inthe x-axis. The sites marked with a diamond are differentiallymethylated sites (FDR<0.05). Dots are estimated OR values, one for eachsite, and the band is the band of union of all the confidence intervalsof 95%. C: control samples, TG: Transgenic.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have developed a method fordiagnosing neurodegenerative diseases based on the determination of themethylation pattern in a sample of mitochondrial DNA from a subject. Theinventors have found that, surprisingly, there are variations in themethylation pattern in the D-loop region and the ND1 gene in subjectssuffering from AD or PD when compared with healthy subjects such asdemonstrated in the examples. Furthermore, the inventors have discovereddifferential methylation patterns associated with the development ofthese diseases.

First Method of the Invention

The first feature of the invention relates to an in vitro method todiagnose or determine the risk of developing a neurodegenerative diseaseselected from Alzheimer's disease and Parkinson's disease in a subject(hereinafter, first method of the invention) that involves determiningin a sample from said subject comprising mitochondrial DNA, themethylation pattern in the D-loop region and/or in the ND1 gene, wherethe meyhylation pattern is determined in at least one site selected fromthe group formed by:

-   -   (i) the CpG sites in the D-loop region shown in Table 1,    -   (ii) the CpG sites in the ND1 gene shown in Table 2;    -   (iii) the CHG sites in the D-loop region shown in Table 3,    -   (iv) the CHG sites in the ND1 gene shown in Table 4, and/or    -   (v) the CHH sites in the D-loop region shown in Table 5;        where there is hypomethylation in at least one of said CpG sites        in the D-loop region, hypermethylation in at least one of said        CHG sites in the D-loop region, hypermethylation in at least one        of said CHH sites in the D-loop region, hypomethylation in at        least one of said CpG sites in the ND1 gene and/or        hypomethylation in at least one of said CHG sites in the ND1        gene, it is indicative that the subject suffers from Alzheimer's        disease or that the subject is at an elevated risk of developing        Alzheimer's disease or where there is hypomethylation in at        least one of said CpG sites in the D-loop region,        hypomethylation in at least one of said CHG sites in the D-loop        region and/or hypomethylation in at least one of the said CHH        sites in the D-loop region, it is indicative that the subject        suffers from Parkinson's disease or that the subject is at an        elevated risk of developing Parkinson's disease.

The term “diagnosis” as used in this document, refers to both theprocess of trying to determine and/or identify a possible disease in asubject, that is to say the diagnostic procedure, as well as the opinionreached through this process, that is to say, the diagnostic opinion. Assuch, it can also be seen as an attempt to classify the status of anindividual in separate and distinct categories that allow medicaldecisions about treatment and prognosis to taken. As will be understoodby the person skilled in the art, such diagnosis may not be correct for100% of the subjects to be diagnosed with, although it is preferred thatit is.

However, the term requires that a statistically significant portion ofsubjects can be identified as suffering from a disease, in particular aneurodegenerative disease selected from Alzheimer's disease andParkinson's disease in the context of the invention, or a predispositionthereto. The person skilled in the art may determine whether a part isstatistically significant using different well known statisticalevaluation tools, for example, by determining confidence intervals,determining the value of p, Student's t-test, the Mann-Whitney test,etc. (See Dowdy Wearden, 1983). Preferred confidence intervals are atleast 50%, at least 60%, at least 70%, at least 80%, at least 90% or atleast 95%. P values are preferably 0.05, 0.025, 0.001 or lower.

The expression “risk of developing a neurodegenerative disease” as usedherein, refers to the predisposition, susceptibility or propensity of asubject to develop a neurodegenerative disease. The risk of developing aneurodegenerative disease generally implies that there is a high or lowrisk or higher or lower risk. Like that, a subject has a high risk ofdeveloping a neurodegenerative disease, particularly Alzheimer's diseaseor Parkinson's disease, has a likelihood of developing this disease ofat least 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90, or at least 95%, or at least 97%, or at least 98%, or at least99%, or at least 100%. Similarly, a subject at a low risk of developinga neurodegenerative disease, particularly Alzheimer's disease orParkinson's disease, is a subject having at least one chance ofdeveloping the disease from at least 0%, or at least one 1%, or at least2%, or at least 3%, or at least 5%, or at least 10%, or at least 20%, orat least 30%, or at least 40%, or at least 49%.

In general, the expression “predict risk”, “risk prediction”, orsimilar, refers to the risk that a patient has of developing aneurodegenerative disease selected from Alzheimer's disease orParkinson's disease, either high or low. As will be understood by theperson skilled in the art, the prediction (or risk), albeit preferred,does not need to be correct for 100% of the subjects to be evaluated,but it is preferable that it is. The term, however, requires that astatistically significant portion of subjects can be identified with ahigher probability to have a particular result. The person skilled inthe art can determine without difficulty whether a part is statisticallysignificant using several well-known statistical tools for assessment,for example, determination of confidence intervals, determination ofp-value, cross-validation classification indices, etc. (more details in“Statistics for research” Dowdy and Wearden, John Wiley & Sons, NewYork, 1983). Preferred confidence intervals are at least 50%, at least60%, at least 70%, at least 80%, at least 90% or at least 95%. P-valuesare, preferably, 0.1, 0.05, 0.02, 0.01 or lower.

The term “neurodegenerative disease” as used herein, includes chronicand progressive processes which are characterized by the selective andsymmetric loss of neurons in motor, sensory and cognitive systems.

The term “Alzheimer's disease” or “senile dementia” or AD refers to amental impairment associated with a specific degenerative brain diseasecharacterized by the appearance of senile plaques, neuritic tangles andprogressive neuronal loss that is clinically manifested in progressivedeficiencies of memory, confusion, behavioral problems, inability tocare for oneself, gradual physical deterioration and, ultimately, death.In preferred embodiments, the Alzheimer's disease at any stage accordingto Braak staging:

-   -   Stages I-II: the brain area affected by the presence of        neurofibrillary tangles corresponding to the transentorhinal        region of the brain    -   Stages III-IV: the affected brain area also extends to areas of        the limbic region such as the hippocampus    -   Stages V-VI: the affected brain area also involves the        neocortical region

This classification by neuropathological stages correlates with theclinical evolution of the existing disease and there is a parallelbetween the decline in memory with the neurofibrillary changes and theformation of neuritic plaques in the entorhinal cortex and thehippocampus (stages I to IV). Also, the isocortical presence of thesechanges (stages V and VI) correlates with clinically severe alterations.The transentorhinal state (I-II) corresponds to clinically silentperiods of disease. The limbic state (III-IV) corresponds to aclinically incipient AD. The necortical state corresponds to a fullydeveloped AD.

The term “Parkinson's disease” or “idiopathic parkinsonism” or“paralysis agitans” or PD as used herein, refers to a chronic,degenerative disease that involves problems of movement control, tremor,rigidity, bradykinesia in all kinds of movements such as walking,sitting, eating, talking, etc., and well as postural instability.Symptoms of the disease are clearly associated with the selectivedegeneration of dopaminergic neurons in the substantia nigra. Thedopaminergic deficit induces a consequent loss of striatal neuronscausing a variety of cytological changes including α-synucleinaggregation in so-called Lewy bodies. The “substantia nigra” is anucleus of the basal ganglia located in the upper portions of themidbrain, under the thalamus and takes its color from the neuromelanin.In preferred embodiments, the PD is in any of the stages according tothe Braak staging:

-   -   Stage I: the affected area is the dorsal motor nucleus and/or        intermediate reticular zone.    -   Stage II: the affected area extends to coreuleus locus and to        the nucleus raphes    -   Stage III: the affected area extends to the midbrain, in        particular the substantia nigra pars compacta.    -   Stage IV: The affected area extends to the transentorhinal        region of the anteromedial temporal mesocortex and alocortex.    -   Stage V: The affected area extends to the insular cortex, the        cingulate cortex and the temporal gyrus.    -   Stage VI: The affected area extends to frontal and parietal area        of the cortex.

The term “subject” as used herein, refers to a person, such as a humanbeing, non-human primate (e.g., chimpanzees and other apes and monkeyspecies), farm animals, such as birds, fish, cattle, sheep, pigs, goatsand horses, Pets such as dogs and cats; mammals, laboratory animalsincluding rodents, such as mice, rats and guinea pigs. The term does notdenote a particular age or sex. In a specific embodiment of theinvention, the subject is a mammal. In a preferred embodiment of theinvention, the subject is a human.

The expression “sample comprising mitochondrial DNA” as used hereinrefers to any sample that can be obtained from a subject in which thereis genetic material from the mitochondria suitable for detecting themethylation pattern.

The term “mitochondrial DNA” or “mtDNA” as used herein, refers to thegenetic material located in the mitochondria of living organisms. It isa closed, circular double-stranded molecule. In humans it consists of16,569 base pairs, containing a small number of genes, distributedbetween the H chain and L chain. Mitochondrial DNA encodes 37 genes: tworibosomal RNA, 22 transfer RNA and 13 proteins that participate inoxidative phosphorylation.

In a specific embodiment of the invention, the sample comprisingmitochondrial DNA is selected from a solid tissue biopsy or biofluid.The samples can be obtained by conventional methods known to the personskilled in the art.

In an even more specific embodiment, the biofluid is selected fromperipheral blood or cerebrospinal fluid.

In an even more specific embodiment, said solid tissue is brain tissue.

In a preferred embodiment of the invention, if desired to diagnose asubject or if desired to determine the risk of developing PD, saidsample is a brain tissue sample obtained from the substantia nigra.

If the material where it is desired to determine the methylation patternaccording to the present method, that is to say the mtDNA, is in a solidtissue or biofluid preferably, it proceeds a prior nucleic acidextraction from the sample using any suitable technique for this. In apreferred embodiment of the invention, the DNA fraction suitable for theimplementation of the invention is 35 total DNA. DNA extraction may becarried out using method known to the persons skilled in the art(Sambrook et al., 2001. “Molecular Cloning: A Laboratory Manual”, 3rded, Cold Spring Harbor Laboratory Press, NY, Vol. 1-3) including,without limitation, density gradient centrifugation, extraction in twostages using aqueous phenol or chloroform with ethanol, columnchromatography, methods based on the ability of DNA to bind itself onglass and/or silicates, such as diatomaceous earth as preparations orcrystal beds, using commercial kits, for example, “Q-Biogene fast DNA @kits” or “QIAamp® (R) DNA Blood Mini Kit” (Qiagen, Hilden, Germany) the“G-Spin IIp” (Intron Biotechnology, Korea) or the “Bio 101 Fast PrepSystem” (Qbiogene®, Madrid, Spain) or the methods described in U.S. Pat.Nos. 5,057,426, 4,923,978 and European Patent Application EP0512767A1.

If desired, the present method can be carried out in samples in whichthe mitochondrial fraction has previously been isolated and subsequentlythe DNA thereof has been isolated. The isolation of the mitochondrialfraction can be carried out using any known method of cellfractionation. Such methods comprise the previous cell disruption bytechniques including physical disruption of the membranes, applicationof ultrasounds, pressure application or enzymatic techniques, followedby differential centrifugation by applying density gradients (such asthose of Ficoll as or Percoll). They can also use commercial kits, forexample, Qproteome “Mitochondrial isolation kit” (Qiagen, Hilden,Germany) or “Mitochondrial isolation kit for cultured cells” (ThermoScientific, USA). These kits are based on the same basic principle,namely cell lysis and differential centrifugation to isolate or enrichthe mitochondrial fraction.

The first method of the invention involves determining the methylationpattern in a sample from a subject comprising mitochondrial DNA. Theterm “DNA methylation” As used herein, refers to a biochemical processinvolving the addition of a methyl group (—CH₃) to DNA cytosinenucleotides (C) or adenine (A). DNA methylation at the 5 position ofcytosine has the specific effect of gene repression and has been foundin all of the vertebrates examined. The term “methylation pattern” asused herein refers but is not limited to the presence or absence ofmethylation of one or more nucleotides. In this way, said one or morenucleotides are comprised in a single nucleic acid molecule.

Said one or more nucleotides are capable of being methylated or not. Theterm “methylation status” can also be used when only considered a singlenucleotide. A methylation pattern can be quantified; in the case it isconsidered more than one nucleic acid molecule.

The term “D-loop” or “control region” as used herein, refers to a regionof non-coding mtDNA containing approximately 1100 base pairs, visibleunder electron microscopy, which is generated during H chain replicationfor the synthesis of a short segment of the heavy strand, 7S DNA.

The term “ND1” or “NADH dehydrogenase 1” or “ND1mt”, as used herein,refers to the gene localized in the mitochondrial genome that encodesthe protein NADH dehydrogenase 1 or ND1. The human ND1 gene sequence isdeposited in the GenBank database (version of Jan. 2, 2014) under theaccession number NC_012920. SEQ ID NO 1. The ND1 protein is part of theenzyme complex called complex I which is active in the mitochondria andis involved in the process of oxidative phosphorylation.

The term “CpG site” as used herein, refers to DNA regions, particularlymitochondrial DNA regions, where a cytosine nucleotide is followed by aguanine nucleotide in linear sequence of bases along its length. “CpG”is an abbreviation for “C-phosphate-G”, i.e., cytosine and guanineseparated by only a phosphate; phosphate binds together any twonucleosides in the DNA. The term “CpG” is used to distinguish thislinear sequence of CG bases pairing of guanine and cytosine. Cytosine inthe CpG dinucleotides may be methylated to form 5-methylcytosine.

The term “CHG site” as used herein, refers to DNA regions, particularlymitochondrial DNA regions, where a cytosine nucleotide and a guaninenucleotide are separated by a variable nucleotide (H) which can beadenine, cytosine or thymine. The cytosine of the CHG site can bemethylated to form 5-methylcytosine.

The term “CHH site” as used herein, refers to DNA regions, particularlyregions of mitochondrial DNA, where a cytosine nucleotide is followed bya first and a second variable nucleotide (H) which can be adenine,cytosine or thymine. The cytosine of the CHG site can be methylated toform 5-methylcytosine.

In a specific embodiment, the first method of the invention comprisesdetermining in a sample of a subject comprising mitochondrial DNA, themethylation pattern in at least one site selected from the CpG sites ofthe D-loop region, selected from the sites shown in Table 1.

TABLE 1 List of CpG positions 16386 and 256 in the D-loop region. CpGsite Position (bp) CpG 2 16427 CpG 3 16449 CpG 4 16454 CpG 5 16495 CpG 616542 CpG 7 16565 CpG 8 33 CpG 9 61 CpG 10 78 CpG 11 80 CpG 12 91 CpG 1396 CpG 14 105 CpG 15 120 CpG 16 162 CpG 17 170 CpG 18 186

The term “determination of the methylation pattern in a CpG site” asused herein, refers to the determination of the methylation status of aparticular CpG site. The determination of the methylation pattern of aCpG site can be performed by multiple processes known to the personskilled in the art.

In a specific embodiment, the first method of the invention involvesdetermining the methylation pattern of at least one CpG site in theD-loop region selected from the sites shown in Table 1. In anotherspecific embodiment, the first method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15 orat least 16 CpG sites selected from Table 1.

In a still more specific and preferred embodiment of the invention, thefirst method of the invention comprises determining the methylationpattern of all of the CpG sites in the D-loop region shown in Table 1.

In another specific embodiment, the first method of the inventioncomprises determining in a sample of a subject containing mitochondrialDNA, the methylation pattern in at least one site selected from CpGsites of the ND1 gene shown in Table 2.

TABLE 2 List of CpG sites in the ND1 gene between the positions 3313 and3686. CpG site Position (bp) CpG 1 3351 CpG 2 3375 CpG 3 3379 CpG 4 3406CpG 7 3453 CpG 12 3549 CpG 13 3642

In another particular embodiment, the first method of the inventioninvolves determining the methylation pattern in a CpG site selected fromthe ND1 gene shown in Table 2. In another specific embodiment, the firstmethod of the invention involves determining the methylation pattern inat least 2, at least 3, at least 4, at least 5 or at least 6 CpG sitesselected from Table 2.

In a still more particular and preferred embodiment of the invention,the first method of the invention involves determining the methylationpattern in all of the CpG sites of the ND1 gene shown in Table 2.

In another particular embodiment, the first method of the inventioncomprises determining in a sample of a subject comprising mitochondrialDNA, the methylation pattern in at least one site selected from the CHGsites in the D-loop region, shown in Table 3.

TABLE 3 List of the CHG sites between positions 16386 and 256 of theD-loop region CHG site Position (bp) CHG 2 16426 CHG 3 16453 CHG 4 16459CHG 5 16466 CHG 6 16479 CHG 7 16514 CHG 8 6 CHG 9 33 CHG 10 64 CHG 11104 CHG 12 122 CHG 13 128 CHG 14 141 CHG 16 253

The term “determination of the methylation pattern in a CHG site” asused herein, refers to the determination of the methylation status of aparticular CHG site.

The determination of the methylation pattern of a CHG site can beperformed by multiple processes known to the person skilled in the art.

In a specific embodiment, the first method of the invention involvesdetermining the methylation pattern of at least one CHG site of theD-loop region selected from the sites shown in Table 3. In anotherspecific embodiment, the first method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13 or at least 14 or at least 15CHG sites selected from Table 3.

In a still more specific and preferred embodiment of the invention, thefirst method of the invention involves determining the methylationpattern in all of the CHG sites of the D-loop region shown in Table 3.

In another specific embodiment, the first method of the inventioncomprises determining in a sample of a subject comprising mitochondrialDNA, the methylation pattern in at least one site selected from CHGsites of the ND1 gene shown in Table 4.

TABLE 4 List of CHG sites in the ND1 gene between the positions 3313 and3686. CHG site Position (bp) CHG 1 3374 CHG 2 3435 CHG 4 3524 CHG 5 3529CHG 6 3589 CHG 7 3641 CHG 8 3657

In another specific embodiment, the first method of the inventioninvolves determining the methylation pattern in at least one CHG site inthe ND1 gene selected from the sites shown in Table 4. In anotherspecific embodiment, the first method of the invention comprisesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5 or at least 6 CHG sites selected from Table 4.

In a still more specific and preferred embodiment of the invention, thefirst method of the invention involves determining the methylationpattern in all of the CHG sites of the ND1 gene shown in Table 4.

In another specific embodiment, the first method of the inventioncomprises determining in a sample of a subject comprising mitochondrialDNA, the methylation pattern in at least one site selected from the CHHsites of the D-loop region, selected from the CHH sites shown in table5.

TABLE 5 List of CHH sites between positions 16386 and 256 positions ofthe D-loop region. CHH site Position (bp) CHH 5 16419 CHH 6 16425 CHH 716429 CHH 8 16439 CHH 9 16442 CHH 10 16446 CHH 11 16451 CHH 12 16458 CHH13 16465 CHH 14 16478 CHH 15 16498 CHH 16 16507 CHH 17 16511 CHH 1816520 CHH 19 16527 CHH 20 16536 CHH 21 16540 CHH 22 16546 CHH 23 16549CHH 24 16560 CHH 25 16563 CHH 26 4 CHH 27 11 CHH 28 15 CHH 29 18 CHH 3026 CHH 31 29 CHH 32 39 CHH 33 43 CHH 34 48 CHH 35 76 CHH 36 86 CHH 37110 CHH 38 113 CHH 39 132 CHH 40 140 CHH 41 144 CHH 42 147 CHH 43 150CHH 44 164 CHH 45 167 CHH 46 190 CHH 47 194 CHH 49 198

The term “determination of a methylation pattern in a CHH site”, as usedherein, refers to determining the methylation status of a particular CHHsite. The determination of the methylation pattern of a CHG site you canbe performed by multiple processes known to the person skilled in theart.

In another specific embodiment, the first method of the inventioncomprises determining the methylation pattern in at least one CHH siteof the D-loop region selected from those shown in Table 5. In anotherspecific embodiment, the first method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28 at least 29, at least 30, at least31, at least 32, at least 33, at least 34, at least 35, at least 36, atleast 37, at least 38, at least 39, at least 40, at least 41, at least42, or at least 43 CHH sites selected from Table 5.

In a still more specific and preferred embodiment of the invention, thefirst method of the invention involves determining the methylationpattern in all of the CHH sites in the D-loop region shown on table 5.

In preferred methods of embodiment, the first method of the inventionincludes:

-   -   (i) determination of the pattern of methylation in all of the        CpG sites of the D-loop region shown in Table 1 and all CpG        sites of the ND1 gene shown in Table 2    -   (ii) determination of the methylation pattern in all of the CpG        sites in the D-loop region shown in Table 1 and in all of the        CHG sites of the D-loop region shown in Table 3    -   (iii) determination of the methylation pattern in all of the CpG        sites of the D-loop region shown in Table 1 and all CHG sites of        the ND1 gene shown in Table 4    -   (iv) determination of the methylation pattern in all CpG sites        in the D-loop region shown in Table 1 and in all CHH sites in        the D-loop region shown in Table 5    -   (v) determination of the methylation pattern in all of the CpG        sites in the ND1 gene shown in Table 2 1 and in all of the CHG        sites in the D-loop region shown on Table 3    -   (vi) determination of the methylation pattern in all of the CpG        sites in the ND1 gene shown in Table 2 and in all of the CHG        sites in the ND1 gene shown in Table 4,    -   (vii) determination of the methylation pattern in all of the CpG        sites in the ND1 gene shown in Table 2 and in all of the CHH        sites of the D-loop region shown in Table 5    -   (viii) determination of the methylation pattern in all of the        CHG sites in the D-loop region shown in Table 3 and all CHG        sites of the ND1 gene shown in Table 4    -   (ix) determination of the methylation pattern in all CHG sites        in the D-loop region shown in Table 3 and/or    -   (x) determination of the methylation pattern in all of the CHG        sites in the ND1 gene shown in Table 4 and in all of the CHH        sites in the D-loop region in Table 5

In some embodiments, the determination of the methylation pattern in atleast one CpG site, at least one CHG site and/or at least one CHH siteaccording to the first method of the invention is carried out in a wholeblood sample, in which case the determination can be made directly. Inother embodiments, the sample that contains mitochondrial DNA,preferably a sample of total DNA, is extracted from cells present in abiological fluid (e.g., whole blood, cerebrospinal fluid) as an initialstage and in such cases, the total nucleic acid extracted from thesesamples represent the suitable work material for the later analysis. TheIsolation of total DNA or mitochondrial DNA can be performed byconventional methods known to the person skilled in the art (citedsupra). After isolating and amplifying (if necessary) the nucleic acidthat contains mitochondrial DNA, the methylation pattern of one or moreCpG sites, one or more CHG sites and/or one or more CHH sites isdetermined. The person skilled in the art will easily recognize that theanalysis of the methylation pattern present in one or several of theCpG, CHG and/or CHH sites described herein in the mitochondrial DNA of asubject, can be carried out by any method or technique capable ofmeasuring the methylation pattern present in such sites.

In another specific embodiment, the first method of the inventioninvolves determining the methylation pattern in said CpG, CHG and/or CHHsites through a technique selected from the group consisting ofMethylation Specific PCR (MSP), a method based on enrichment. (e.g.MeDIP, MBD-seq and MethylCap), bisulfite sequencing and through abisulphite based method (eg. RRBs, Infinium, GoldenGate, Cobra, MSP,MethyLight) and a method by restriction of digestion (e.g., MRE-seq, orHELP trial), pyrosequencing, or differential conversion, differentialrestriction, differential weight of the methylated DNA CpG, CHG and/orCHH sites.

In a specific and preferred embodiment of the invention the methylationpattern of one or more CpG, CHG and/or CHH sites in the D-loop regionand/or one or more CpG and/or CHG sites in the ND1 gene is determinedthrough pyrosequencing. Briefly, this technique is based on theprinciple of sequencing by synthesis and detection of releasedpyrophosphate (PPi) during DNA synthesis. This technique employs aseries of four enzymes to detect nucleic acid sequences during thesynthesis process; DNA polymerase, ATP sulfurylase, luciferase, apyraseand adenosine 5′ fosofosulphate (APS) and luciferin used as substrates.

To determine the methylation pattern in mitochondrial DNA, it isnecessary to chemically treat said sample so that all cytosineunmethylated bases are modified at uracil bases, or another base whichdiffers from cytosine in terms of base pairing behavior, while the basesof 5 methylcytosine remain unchanged. The term “modify” as used hereinmeans the conversion of an unmethylated cytosine to another nucleotidethat will distinguish the unmethylated cytosine from the methylatedcytosine. The conversion of unmethylated cytosine bases, but notmethylated, in the sample containing mitochondrial DNA is carried outwith a conversion agent. The term “conversion agent” or “conversionreagent” as used herein, refers to a reagent capable of converting anunmethylated cytosine to uracil or another base that is differentiallydetectable to cytosine in terms of hybridization properties. Theconversion agent is preferably a bisulfate such as bisulfites orhydrogen sulfite. However, other agents that similarly modifyunmethylated cytosine, but not methylated cytosine can also be used inthis method of the invention, such as hydrogen sulfite. The reaction isperformed according to standard procedures (Frommer et al., 1992, Proc.Natl. Acad. Sci. USA 89: 1827-1831; Olek, 1996, Nucleic Acids Res. 24:5064-6; EP 1394172). It is also possible to carry out the conversionenzymatically, e.g. using cytidine deaminases specific methylation.

In a preferred embodiment of the first method of the invention, thesample containing mitochondrial DNA has been treated with a reagentcapable of converting an unmethylated cytosine to uracil or another basethat is detectably different from cytosine in terms of hybridizationproperties. In a more preferable embodiment, the sample comprisingmitochondrial DNA is treated with bisulfite using an appropriatecommercial kit, for example “EZ Methylation Kit” (Zymo Research, Ecogen;Barcelona, Spain).

Once the sample containing mitochondrial DNA has been treated with abisulfite, the D-loop region and/or the ND1 gene containing one or moreCpG, CHG and/or CHH sites shown in Tables 1 to 5 can be amplified usingprimers that distinguish unmethylated sequence (in which the cytosine ofthe CpG site is converted into uracil) from the methylated sequence (inwhich the cytosine in the CpG site remains as cytosine). Manyamplification methods rely on an enzymatic chain reaction such as, forexample, a polymerase chain reaction (PCR), ligase chain reaction (LCR),ligase chain reaction Polymerase 35, Gap-LCR, repair chain reaction, 3SRand NASBA. Furthermore, there is strand displacement amplification(SDA), transcription mediated amplification (TMA), and Qβ-amplification,etc, this being merely an illustrative list. Methods of nucleic acidamplification are described in Sambrook et al., 2001 (cited supra).Other amplification methods include PCR method specific methylation(MSP) described in U.S. Pat. No. 5,786,146 which combines bisulfitetreatment and allele-specific PCR (see, e.g., U.S. Pat. Nos. 5,137,806,5,595,890, 5,639,611). Uracil is recognized as a thymine by Taqpolymerase and therefore, according to PCR, the resultant productcontains cytosine only at the position where 5-methylcytosine DNA existin the starting template.

In a preferred embodiment of the invention, once the sample comprisingmitochondrial DNA, preferably a sample of total DNA, has been treatedwith bisulfite, the region containing one or more CpG, CHG and/or CHHsite(s) can be amplified using primers that are not specific to themethylated sequence. For example, the preferred sequence of the primersnot corresponding to a nucleotide sequence comprising a CpGdinucleotide.

Amplification products are detected according to standard procedures inprior art. The amplified nucleic acid can be determined through methodsknown to person skilled in the art and are described in e.g., Sambrooket al., 2001 (cited et supra). There may also be additional purificationsteps before the target nucleic acid is detected, for example aprecipitation step. The detection methods may include but may notlimited to binding or interleaving of specific dyes such as ethidiumbromide which intercalate into double stranded DNA and changes itsfluorescence thereafter. The purified nucleic acids may also beseparated by electrophoretic methods optionally after a restriction ofdigestion and be displayed later. There are also probe-based assayswhich exploit the oligonucleotide hybridization to specific sequencesand subsequent detection of the hybrid. It is also possible to sequencethe target nucleic acid after further steps known to the person skilledin the art. Other methods use various nucleic acid sequences with asilicon chip to which specific probes are bound and produce a signalwhen a complementary sequence binds.

In a preferred embodiment of the invention, after amplification of theregion of interest where it is desired to determine the methylationpattern (eg in the D-loop region or in the ND1 gene) pyrosequencing isused to determine in said sequence CpG, CHG and/or modified CHH sitesafter treatment with bisulphite The reason for cytosine/thymine in eachof the sites can be quantitatively determined based on the amount ofcytosine and thymine incorporated during the extension step of thesequence.

Alternatively, the methylation pattern of at least one CpG, CHG and/orCHH sites in the D-loop region or in at least one CpG and/or CHG site ofthe ND1 gene of the site can be confirmed by restriction enzymedigestion and Southern blot analysis. Examples of endonucleasesmethylation sensitive restriction that may be used include SmaI, SacII,EagI, MspI, HpaII, BstUIY BssHII, for example.

The term “hypermethylation” as used herein, refers to an alteredmethylation pattern where one or more nucleotides, preferably cytosinesfrom CpG, CHG and/or CHH sites are methylated compared to a referencesample. Said reference sample is preferably a sample that containsmitochondrial DNA obtained from a subject not suffering from aneurodegenerative disease selected from AD or PD. In particular, theterm refers to a number larger than 5-methylcytosines in one or more CpGsites in the D-loop region shown in Table 1, in one or more CpG sites inthe ND1 gene shown in Table 2, in one or more CHG sites in the D-loopregion shown in Table 3, in one or more CHG sites in the ND1 gene shownin Table 4 and/or one or more CHH sites in the D-loop region shown inTable 5, in a sequence of mitochondrial DNA when compared with therelative amount of 5-methylcytosines present in said one or more sitesin a reference sample.

The term “hypomethylation” as used herein, refers to an alteredmethylation pattern where one or more nucleotides, preferably cytosinesin the CpG, CHG and/or CHH sites, are unmethylated compared to areference sample. The term “reference sample” refers to a samplecontaining mitochondrial DNA obtained from a subject not suffering froma neurodegenerative disease selected from AD or PD. In particular, saidterm refers to a small number of 5-methylcytosines in one or more CpGsites in the D-loop region shown in Table 1, in one or more CpG sites ofthe ND1 gene shown in Table 2, in one or more sites CHG sites in theD-loop region shown in Table 3, in one or more CHG sites in the ND1 geneshown in Table 4 and/or one or more CHH sites in the D-loop region shownin Table 5 in a sequence of mitochondrial DNA as compared to therelative amount of 5-methylcytosines present in said one or more CpGsites, one or more CHG sites and/or one or more CHH sites in a referencesample.

In a preferred embodiment of the invention, said reference samplecontaining mitochondrial DNA is selected from tissue samples, orbiofluids, preferably blood samples or cerebrospinal fluid of subjects.In a preferred embodiment, said reference sample is total DNA. Methodsto obtain these samples as well as methods of isolating total DNA ormitochondrial DNA of a sample have been detailed above. In a still morepreferred embodiment, the reference sample is a sample containingmitochondrial DNA from age-matched subjects.

In this first method, the invention provides some specific CpG, CHH andCHG sites that are related to the diagnosis or risk of developing aneurodegenerative disease selected from Alzheimer's disease andParkinson's disease. Therefore:

-   -   hypermethylation in at least one of the CpG sites in the D-loop        region shown in Table 1,    -   hypermethylation in at least one of the CHG sites in the D-loop        region shown in Table 3,    -   hypermethylation in at least one of the CHH sites in the D-loop        region shown in Table 5,    -   hypomethylation in at least one of the CpG sites in the ND1 gene        shown in Table 2, and/or    -   hypomethylation in at least one of the CHG sites in the ND1 gene        shown in Table 4, is indicative that the subject suffers from        Alzheimer's disease or that the subject is at a high risk of        developing Alzheimer's disease; or    -   hypomethylation in at least one of the CpG sites in the D-loop        region shown in Table 1,    -   hypomethylation in at least one of the CHG sites in the D-loop        region shown in Table 3, and/or    -   hypomethylation in at least one of the CHH sites in the D-loop        region shown in Table 5 is indicative that the subject suffers        from Parkinson's or that the subject is at a high risk of        developing Parkinson's disease.

In a specific embodiment, the first method of the invention involvesdetermining the methylation pattern of all the CpG sites, all CHG sitesand all CHH sites in the D-loop region shown in Tables 1, 3 and 5, andthe methylation pattern of all the CpG sites all of all CHG sites of theND1 gene shown in Tables 2 and 4.

The authors of the present invention have found that the degree ofmethylation in the CpG, CHG and CHH sites in the D-loop region isgreater in subjects suffering from Alzheimer's in stages I-II than insubjects suffering from the disease in stages III-IV.

In a specific embodiment, if the methylation pattern with respect to thereference pattern is observed in a sample containing mitochondrial DNAfrom a subject diagnosed with Alzheimer's disease stage I-II, thenhypomethylation in at least one of the CpG sites in the D-loop regionshown in Table 1 or hypomethylation in one of said CHG sites in theD-loop region shown in Table 3, indicates that the subject suffers fromAlzheimer's disease stage III-IV.

Second Method of the Invention

In a second aspect, the invention relates to an in vitro method toselect a subject to be submitted to preventive treatment of aneurodegenerative disease selected from Alzheimer's disease andParkinson's disease in a subject (hereinafter, second method of theinvention) that involves determining in a sample of said subjectcontaining mitochondrial DNA the methylation pattern in the D-loopregion, and/or in the ND1 gene, where the methylation pattern isdetermined in at least one site selected from the group formed by:

-   -   i. the CpG sites in the D-loop region shown in Table 1,    -   ii. the CpG sites in the ND1 gene shown in Table 2;    -   iii. the CHG sites in the D-loop region shown in Table 3,    -   iv. the CHG sites in the ND1 gene shown in Table 4, and/or    -   v. the CHH sites in the D-loop region shown in Table 5        wherein a hypermethylation in at least one of said CpG sites in        the D-loop region, a hypermethylation in at least one of said        CHG sites in the D-loop region, a hypermethylation in at least        one of said CHH sites in the D-loop region, a hypomethylation in        at least one of said CpG sites in the ND1 gene and/or a        hypomethylation in at least one of said CHG sites in the ND1        gene is indicative that the subject is a candidate to receive        treatment aimed at preventing Alzheimer's disease or        wherein a hypomethylation in at least one of said CpG sites in        the D-loop region, a hypomethylation in at least one of said CHG        sites in the D-loop region and/or a hypomethylation in at least        one of the said CHH sites in the D-loop region it is indicative        that the subject is a candidate to receive treatment aimed at        preventing Parkinson's disease.

The term “preventive treatment”, as used herein, refers to theprevention or conjunction of prophylactic measures to prevent disease toprevent or delay the onset of symptoms thereof as well as reducing oralleviating clinical symptoms thereof. In particular, the term refers tothe prevention or set of measures to prevent the occurrence, to delay orto relieve the clinical symptoms associated with a neurodegenerativedisease selected from Alzheimer's and Parkinson's disease. Desiredclinical outcomes associated with the administration of the treatment toa subject include but are not limited to, stabilization of thepathological stage of the disease, delay in the progression of thedisease or improvement in the physiological state of the subject.

Suitable preventative treatments aimed at preventing or delaying theonset of the symptoms of Alzheimer's disease include but are not limitedto, cholinesterase inhibitors such as donepezil hydrochloride (Arecept),rivastigmine (Exelon) and galantemina (Reminyl) or antagonistsN-methyl-D-aspartate (NMDA). Treatments aimed at preventing or delayingthe onset of the symptoms of Parkinson's disease include but are notlimited to, L-dopa, inhibitors of catechol-o-methyl transferase (COMT)such as tolcapone (Tasmar) and entacapone (Comtan), monoamine oxidase B(MAOB) such as selegiline (Eldepryl) and rasagaline (Azilect) anddopamine agonists such as pramipexole, rotigotine and ropinirole.

The term “to select” as used herein, refers to the action of choosing asubject for submission to preventive treatment of a neurodegenerativedisease selected from Alzheimer's disease and Parkinson's disease.

The terms “subject”, “neurodegenerative disease”, “Alzheimer's disease”,“Parkinson's disease”, “sample”, “mitochondrial DNA”, “D-loop region”,“ND1 gene”, “CpG site”, “CHG site”, CHH site”, “methylation pattern”,“hypermethylation” and “hypomethylation” have been described in detailin the context of the first method of the invention and used with thesame meaning in the second method of the invention.

In a specific embodiment of the second method of the invention, thesample comprising mitochondrial DNA is selected from a biopsy of a solidtissue or biofluid. The samples can be obtained by conventional methodsknown to the person skilled in the art.

In an even more specific embodiment, the biofluid is selected fromperipheral blood or cerebrospinal fluid.

In an even more specific embodiment, said solid tissue is brain tissue.In a preferred embodiment of the invention, if desired to select asubject to be submitted to preventive treatment of Parkinson's disease,said sample is a brain tissue sample obtained from the substantia nigra.

In a specific embodiment, the second method of the invention involvesdetermining in a sample of a subject containing mitochondrial DNA, themethylation pattern in at least one site selected from CpG sites in theD-loop region, shown in Table 1.

In a specific embodiment, the second method of the invention involvesthe determination of the methylation pattern in at least one CpG site inthe D-loop region selected from those shown in Table 1. In anotherspecific embodiment, the first method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15 orat least 16 CpG sites selected from Table 1.

In a still more specific and preferred embodiment of the invention, thesecond method of the invention involves determining the methylationpattern in all of the CpG sites in the D-loop region shown in Table 1.

In another specific embodiment, the second method of the inventioninvolves determining in a sample of a subject comprising mitochondrialDNA, the methylation pattern in at least one site selected from the CpGsites in the ND1 gene, shown in Table 2.

In another specific embodiment, the second method of the inventioninvolves determining the methylation pattern in a CpG site in the ND1gene selected from the sites shown in Table 2. In another specificembodiment, the second method of the invention involves determining themethylation pattern in at least 2, at least 3, at least 4, at least 5 orat least 6 CpG sites selected from Table 2.

In a still more specific and preferred embodiment of the invention, thesecond method of the invention involves determining the methylationpattern of all of the CpG sites in the ND1 gene shown in Table 2.

In another particular embodiment, the second method of the inventioninvolves determining in a sample of a subject containing mitochondrialDNA, the methylation pattern in at least one site selected from the CHGsites in the D-loop region, shown in Table 3.

In a particular embodiment, the second method of the invention involvesdetermining the methylation pattern in at least one CHG site in theD-loop region selected from the sites shown in Table 3. In anotherspecific embodiment, the second method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13 or at least 14 or at least 15CHG sites selected from Table 3.

In a still more specific and preferred embodiment of the invention, thesecond method of the invention involves determining the methylationpattern of all the CHG sites in the D-loop region shown in Table 3.

In another specific embodiment, the second method of the inventioninvolves the determination in a sample of a subject that containsmitochondrial DNA, the methylation pattern in at least one site selectedfrom the CHG sites in ND1 gene, shown in Table 4.

In another specific embodiment, the second method of the inventioninvolves determining the methylation pattern in at least one CHG site inthe ND1 gene selected from the sites shown in Table 4. In anotherspecific embodiment, the second method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5 or at least 6 CpG sites selected from Table 4.

In a still more particular and preferred embodiment of the invention,the second method of the invention involves determining the methylationpattern in all of the CHG sites of the ND1 gene shown in Table 4.

In another specific embodiment, the second method of the inventioninvolves determining in a sample of a subject containing mitochondrialDNA, the methylation pattern in at least one site selected from the CHHsites in the D-loop region, shown in Table 5.

In another specific embodiment, the second method of the inventioninvolves determining the methylation pattern in at least one CHH site inthe D-loop region selected from those shown in Table 5. In anotherspecific embodiment, the second method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 30, at least31, at least 32, at least 33, at least 34, at least 35, at least 36, atleast 37, at least 38, at least 39 at least 40, at least 41, at least 42or at least 43 CHH sites selected from Table 5.

In a still more specific and preferred embodiment of the invention, thesecond method of the invention involves determining the methylationpattern in all of the CHH sites in the D-loop region shown in Table 5.

In preferred methods of embodiment, the second method of the inventionincludes:

-   -   (i) determination of the methylation pattern in all of the CpG        sites in the D-loop region shown in Table 1 and in all of the        CpG sites in the ND1 gene shown in Table 2    -   (ii) determination of the methylation pattern in all of the CpG        sites in the D-loop region shown in Table 1 and in all of the        CHG sites in the D-loop region shown in Table 3    -   (iii) determination of the methylation pattern in all of the CpG        sites in the D-loop region shown in Table 1 and in all of the        CHG sites in the ND1 gene shown in Table 4    -   (iv) determination of the methylation pattern in all CpG sites        in the D-loop region shown in Table 1 and in all CHH sites in        the D-loop region shown in Table 5    -   (v) determination of the methylation pattern in all of the CpG        sites in the ND1 gene shown in Table 2 1 and in all CHG sites in        the D-loop region shown in Table 3    -   (vi) determination of the methylation pattern in all of the CpG        sites in the ND1 gene shown in Table 2 and in all of the CHG        sites in the ND1 gene shown in Table 4    -   (vii) determination of the methylation pattern in all CpG sites        in the ND1 gene shown in Table 2 and in all the CHH sites in the        D-loop region shown in Table 5    -   (viii) determination of the methylation pattern in all of the        CHG sites in the D-loop region shown in Table 3 and in all of        the CHG sites in the ND1 gene shown in Table 4    -   (ix) determination of the methylation pattern in all of the CHG        sites in the D-loop region shown in Table 3 and/or    -   (x) determination of the methylation pattern in all of the CHG        sites in the ND1 gene shown in Table 4 and in all of the CHH        sites in the D-loop region shown in Table 5

Suitable methods for determining the methylation pattern in a sample ofa subject containing mitochondrial DNA have been described in detail inthe context of the first method of the invention.

In a specific embodiment, the second method of the invention involvesdetermining the methylation pattern in said CpG, CHG and/or CHH sitesthrough a technique selected from the group consisting ofMethylation-Specific PCR-based enrichment method (e.g. MeDIP, MBD-seqand MethylCap), sequencing bisulfite and based on bisulfite method (e.g.RRBs, Infinium, GoldenGate, Cobra, MSP, MethyLight) and a method forrestriction digestion (e.g., MRE-seq, or HELP trial), pyrosequencing,assay chiP-on-chip, or differential conversion, differentialrestriction, differential weight of the CpG, CHG and/or CHH sitesmethylated DNA.

In a specific and preferred embodiment of the invention the methylationpattern of one or more CpG, CHG and/or CHH sites in the D-loop regionand/or one or more CpG or CHH sites in the ND1 gene, according to thesecond method of the invention is determined by pyrosequencing.

According to the second method of the invention:

-   -   hypermethylation in at least one of the CpG sites in the D-loop        region shown in Table 1,    -   hypermethylation in at least one of CHG sites in the D-loop        region shown in Table 3,    -   hypermethylation in at least one of the CHH sites in the D-loop        region shown in Table 5    -   hypomethylation in at least one of the CpG sites in the ND1 gene        shown in Table 2    -   and/or hypomethylation in at least one of the CHG sites in the        ND1 gene shown in Table 4, is indicative that the subject is a        candidate to receive treatment aimed at preventing Alzheimer's        disease; or    -   hypomethylation in at least one of the CpG sites in the D-loop        region shown in Table 1    -   hypomethylation in at least one of the CHG sites in the D-loop        region shown in Table 3,    -   and/or hypomethylation in at least one of the CHH sites in the        D-loop region shown in Table 5        is indicative that the subject is a candidate to receive        treatment aimed at preventing Parkinson's disease.

In a specific embodiment, the second method of the invention involvesdetermining the methylation pattern of all the CpG sites, all CHG sitesand all of the CHH sites in the D-loop region shown in Tables 1, 3 and5, and the methylation pattern of all the CpG sites and all of the CHGsites in the ND1 gene shown in Tables 2 and 4.

Third Method of the Invention

In the third feature, the invention relates to an in vitro method tomonitor the progression of a neurodegenerative disease selected fromAlzheimer's or Parkinson's disease in a subject (hereinafter, thirdmethod of the invention) Involving:

-   -   (a) Determination in a sample from said subject containing        mitochondrial DNA, the methylation pattern in the D-loop region,        and/or in the ND1 gene, where the methylation pattern is        determined at least one site selected from the group formed by:        -   (i) the CpG sites in the D-loop region shown in Table 1,        -   (ii) the CpG sites in the ND1 gene shown in Table 2;        -   (iii) the CHG sites in the D-loop region shown in Table 3,        -   (iv) the CHG sites in the ND1 gene shown in Table 4, and/or        -   (v) the CHH sites in the D-loop region shown in Table 5 and    -   (b) comparing the methylation pattern determined in step a) with        said methylation pattern obtained in an earlier stage of the        disease, in the case of hypomethylation in at least one of said        CpG sites in the D-loop region, hypomethylation in at least one        of said CHG sites in the D-loop region, hypomethylation in at        least one of the said CHH sites in the D-loop region,        hypomethylation in at least one of said CpG sites in the ND1        gene and/or hypomethylation in at least one of said CHG sites in        the ND1 gene relative to said methylation pattern determined at        an earlier stage of disease, is indicative of the advance of        Alzheimer's disease; and where there is hypomethylation in at        least one of said CpG sites in the D-loop region,        hypomethylation in at least one of said CHG sites in the D-loop        region and/or hypomethylation in at least one of said CHH sites        in the D-loop region with respect to said methylation pattern        determined at an earlier stage of the disease is indicative of        the advance of Parkinson's disease.

The term “monitor the progression” which is equivalent to “determine theprognosis” refers to determining the progression of a disease in asubject diagnosed with said disease. Particularly, the term refers todetermining the progression of a neurodegenerative disease selected fromAlzheimer's disease and Parkinson's disease in a subject diagnosed withsaid disease. As the person skilled in the art knows, there are severalsuitable parameters to determine the evolution of a disease in asubject, for example, the evolution of a neurodegenerative diseaseselected from AD and PD, can be determined by, determination of overallsurvival.

In a specific embodiment, the subject under study has been diagnosedwith AD stage I-II.

In another specific embodiment, the subject under study has beendiagnosed with PD in stages III-V.

The term “overall survival”, as used herein, refers to the percentage ofpatients who survive from the time of diagnosis or treatment of aneurodegenerative disease selected from Alzheimer's and Parkinson'sdisease, after a defined period of time.

The terms “subject”, “neurodegenerative disease”, “Alzheimer's disease”,“Parkinson's disease”, “sample”, “mitochondrial DNA”, “D-loop region”,“ND1 gene”, “CpG site”, “CHG site”, “CHH site”, “methylation pattern”,“hypermethylation” and “hypomethylation” have been described in detailin the context of the first method of the invention and are used withthe same meaning in the third method of the invention.

According to the third method of the invention, the first step ofdetermining the prognosis of a neurodegenerative disease selected fromAD and PD involves determining in a sample containing mitochondrial DNAfrom a subject diagnosed with said neurodegenerative disease, themethylation pattern in the D-loop region and/or in the ND1 gene in atleast one site selected from sites shown in Tables 1 to 5.

In a second step, the third method of the invention involves comparingthe methylation pattern obtained in said first step with saidmethylation pattern obtained in an earlier stage of disease. Therefore,the third method of the invention involves determining the methylationpattern in a sample containing mitochondrial DNA (first sample) from asubject diagnosed with said neurodegenerative disease, the methylationpattern in the D-loop region and/or in the ND1 gene in at least one siteselected from the CpG, CHG and/or CHH sites shown in Tables 1 to 5 and,after a suitable period of time, determining in a sample containingmitochondrial DNA (second sample) of said subject diagnosed with saidneurodegenerative disease, the methylation pattern at these sites. Saidsecond sample can be obtained a period of one month, two months, threemonths, four months, five months, six months, one year, two years, threeyears, four years, five years, ten years or more after obtaining thefirst sample.

In a particular embodiment, said first sample is obtained from a subjectwho is not receiving any proper treatment for said neurodegenerativedisease selected from AD and PD and said second sample is obtained aftera period of time where treatment of the disease is taking place. Inanother specific embodiment, said first sample is obtained at thebeginning of appropriate treatment for said neurodegenerative diseaseand the second sample is obtained at one or more points during thecourse of the treatment.

According to the third method of the invention:

-   -   hypomethylation in at least one of the CpG sites in the D-loop        region shown in Table 1,    -   hypomethylation in at least one of the CHG sites in the D-loop        region shown in Table 3,    -   hypomethylation in at least one of the CHH sites in the D-loop        region shown in Table 5,    -   hypomethylation in at least one of the CpG sites in the ND1 gene        shown in Table 2,    -   and/or hypomethylation in at least one of the CHG sites in the        ND1 gene shown in Table 4, with respect to said methylation        pattern determined at an earlier stage of disease is indicative        of the advance of Alzheimer's disease; or    -   hypomethylation in at least one of the CpG sites in the D-loop        region shown in Table 1,    -   hypomethylation in at least, one of the CHG sites in the D-loop        region shown in Table 3,    -   and/or hypomethylation in at least one of the CHH sites in the        D-loop region shown in Table 5,        with respect to said methylation pattern determined at an        earlier stage of disease is indicative of the advance of        Parkinson's disease.

The expression “advance of Alzheimer's disease” as used herein, refersto the subject being at a more evolved stage of the disease with respectto the stage where the subject was diagnosed. That is, if the subjectwas classified as stage I-II of the AD (according to the brainimpairment and/or symptoms or clinical manifestations of the diseasepresent in that subject) it is considered that the subject is at a moreadvanced stage of the disease if the subject goes from being classifiedat stage III-IV or a stage V-VI or if the subject goes from beingclassified as stage II I-IV to being classified as stage V-VI stage ofAD.

The expression “advancement of Parkinson's disease” as used herein,refers to the subject being at a more evolved stage of the disease withrespect to the stage where the subject was diagnosed. That is, if thesubject was classified as stage I-II of PD (according to brainimpairment and/or symptoms or clinical manifestations of disease presentin that subject) it is considered that the subject is in a more advancedstage of the disease if the subject goes from being classified as stageIII-IV or at stage V-VI or if the subject goes from being classified asstage III-IV to being classified as stage V-VI of PD.

In a specific embodiment of the third method of the invention, thesample comprising mitochondrial DNA is selected from a biopsy of a solidtissue or biofluid. The samples can be obtained by conventional methodsknown to the persons skilled in the art

In an even more specific embodiment, the biofluid is selected fromperipheral blood or cerebrospinal fluid.

In an even more specific embodiment, said solid tissue is brain tissue.

In a preferred embodiment of the invention, if the progression ofParkinson's disease is monitored, said brain tissue sample is obtainedfrom the substantia nigra.

In a particular embodiment, the third method of the invention involvesdetermining in a sample of a subject containing mitochondrial DNA, themethylation pattern in at least one site selected from the CpG sites inthe D-loop region, shown in table 1.

In a specific embodiment, the third method of the invention involvesdetermining the methylation pattern of at least one CpG site in theD-loop region selected from the sites shown in Table 1. In anotherspecific embodiment, the first method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15 orat least 16 CpG sites selected from Table 1.

In a still more specific and preferred embodiment of the invention, thethird method of the invention comprises determining the methylationpattern of all of the CpG sites in the D-loop region shown in Table 1.

In another specific embodiment, the third method of the inventioncomprises determining in a sample of a subject containing mitochondrialDNA, the methylation pattern in at least one site selected from the CpGsites in the ND1 gene shown in Table 2.

In another specific embodiment, the third method of the inventioninvolves determining the methylation pattern in a CpG site in the ND1gene selected from the sites shown in Table 2. In another specificembodiment, the third method of the invention involves determining themethylation pattern in at least 2, at least 3, at least 4, at least 5 orat least 6 CpG sites selected from Table 2.

In a still more specific and preferred embodiment of the invention, thethird method of the invention involves determining the methylationpattern in all of the CpG sites in the ND1 gene shown in Table 2.

In a specific embodiment, the third method of the invention involvesdetermining in a sample of a subject containing mitochondrial DNA, themethylation pattern in at least one site selected from the CHG sites inthe D-loop region, shown in Table 3.

In a specific embodiment, the third method of the invention involvesdetermining the methylation pattern in at least one CHG site in theD-loop region selected from the sites shown in Table 3. In anotherspecific embodiment, the third method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13 or at least 14 or 15 CHG sitesselected from Table 3.

In a still more specific and preferred embodiment of the invention, thethird method of the invention involves determining the methylationpattern in all of the CHG sites in the D-loop region shown in Table 3.

In another specific embodiment, the third method of the inventioninvolves determining in a sample of a subject containing mitochondrialDNA, the methylation pattern in at least one site selected from the CHGsites in the ND1 gene, shown in Table 4.

In another specific embodiment, the third method of the inventioninvolves determining the methylation pattern in at least one CHG site inthe ND1 gene selected from the sites shown in Table 4. In anotherspecific embodiment, the third method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5, or at least 6 CpG sites selected from Table 4.

In a still more specific and preferred embodiment of the invention, thethird method of the invention involves determining the methylationpattern in all of the CHG sites of the ND1 gene shown in Table 4.

In another specific embodiment, the third method of the inventioninvolves determining in a sample of a subject containing mitochondrialDNA, the methylation pattern in at least one site selected from the CHHsites in the D-loop region, shown in Table 5.

In another specific embodiment, the third method of the inventioninvolves determining the methylation pattern in at least one CHH site inthe D-loop region selected from the sites shown in Table 5. In anotherspecific embodiment, the third method of the invention involvesdetermining the methylation pattern in at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40, at least 41, at least 42, or atleast 43 CHH sites selected from Table 5.

In a still more specific and preferred embodiment of the invention, thethird method of the invention involves determining the methylationpattern in all of the CHH sites in the D-loop region shown in Table 5.

In preferred methods of embodiment, the third method of the inventionincludes:

i. determination of the methylation pattern in all of the CpG sites inthe D-loop region shown in Table 1 and in all of the CpG sites in theND1 gene shown in Table 2

ii. determination of the methylation pattern in all of the CpG sites inthe D-loop region shown in Table 1 and in all CHG sites of the D-loopregion shown in Table 3,

iii. determination of the methylation pattern in all of the CpG sites inthe D-loop region shown in Table 1 and in all of the CHG sites in theND1 gene shown in Table 4,

iv. determination of the methylation pattern in all CpG sites in theD-loop region shown in Table 1 and in all CHH sites in the D-loop regionshown in Table 5,

v. determination of the methylation pattern in all CpG sites of the ND1gene shown in Table 2 1 and in all the CHG sites in the D-loop regionshown in Table 3,

vi. determination of the methylation pattern in all of the CpG sites inthe ND1 gene shown in Table 2 and in all of the CHG sites in the ND1 30gene shown in Table 4,

vii. determination of the methylation pattern in all CpG sites in theND1 gene shown in Table 2 and in all the CHH sites in the D-loop regionshown in Table 5.

viii. determination of the methylation pattern in all of the CHG sitesin the D-loop region shown in Table 3 and all of the CHG sites in theND1 gene shown in Table 4 and/or

ix. determination of the methylation pattern in all of the CHG sites inthe D-loop region shown in Table 3

x. determination of the methylation pattern in all CHG sites in the ND1gene shown in Table 4 and in all the CHH sites in the D-loop regionshown in Table 5.

Suitable methods to determine the methylation pattern in a sample of asubject containing mitochondrial DNA methods have been described indetail in the context of the first method of the invention.

In another specific embodiment, the third method of the inventioninvolves determining the methylation pattern in said CpG, CHG and/or CHHsites through a technique selected from the group consisting ofMethylation Specific PCR (MSP), a method based on enrichment. (e.g.MeDIP, MBD-seq and MethylCap), bisulfite sequencing and through abisulphite based method (eg. RRBs, Infinium, GoldenGate, Cobra, MSP,MethyLight) and a method by restriction of digestion (e.g., MRE-seq, orHELP trial), pyrosequencing, or differential conversion, differentialrestriction, differential weight of the methylated DNA CpG, CHG and/orCHH sites.

In a specific and preferred embodiment of the invention the methylationpattern of one or more CpG, CHG and/or CHH sites in the D-loop regionand/or one or more CpG or CHH sites in the ND1 gene, according to thethird method of the invention, is determined by pyrosequencing.

In another specific embodiment, the third method of the inventioninvolves determining the methylation pattern of all CpG sites, all CHGsites and all CHH sites in the D-loop region shown in Tables 1, 3 and 5,and the methylation pattern of all CpG sites and all of all the CHGsites in the ND1 gene shown in Tables 2 and 4.

Fourth Method of the Invention

The authors of the present invention have discovered a single nucleotidepolymorphism (SNP) in the D-loop region of mitochondrial DNA that isstatistically associated with the development of Alzheimer's disease ifthe SNP is in at least 60% of the mtDNA molecules of a subject.

Therefore, in the fourth feature, the invention relates to an in vitromethod to diagnose or determine the risk of developing Alzheimer'sdisease in a subject (hereinafter, the fourth method of the invention)that involves determining in a sample that contains mitochondrial DNA ofsaid subject the nucleotide at the polymorphic position 16519 accordingto the sequence defined under accession number NC_012920 (SEQ ID NO: 1)in the NCBI database, where detection of the nucleotide C at saidpolymorphic position or the presence of the nucleotide C at saidpolymorphic position in at least 60% of the mitochondrial DNA moleculesof said subject is indicative that the subject suffers from said diseaseor that the subject has an elevated risk of developing the disease.

The terms “diagnosis”, “determine the risk”, “sample” and “mitochondrialDNA” have been defined in the context of the first, second and thirdmethod of the invention and are used with the same meaning in the fourthmethod of the invention.

The presence of a specific nucleotide at a polymorphic position can bedefined as the percentage of DNA molecules having such nucleotide atsaid polymorphic position with respect to the total of DNA moleculespresent in the sample. According to the fourth method of the inventionit is considered that the subject is suffering from Alzheimer's diseaseor that the subject has an elevated risk of developing said disease ifthe mtDNA of said subject presents the nucleotide C at the polymorphicposition 16519 according to the sequence defined under the accessionnumber NC_012920 when the percentage of molecules that present saidnucleotide at that position is at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90% or more.

In a specific embodiment, it is considered that the subject hasAlzheimer's disease or that the subject has an elevated risk ofdeveloping the disease if the MtDNA of said subject presents thenucleotide C at the polymorphic position 16519 according to the sequencedefined under the accession number NC_012920 in 60% of mtDNA molecules.In a preferred embodiment of the invention it is considered that thesubject has Alzheimer's disease or that the subject has an elevated riskof developing said disease if the MtDNA of said subject presents thenucleotide C at the polymorphic position 16519 according to the sequencedefined under the accession number NC_012920 in 71% of mtDNA molecules.

In another preferred embodiment of the invention, it is considered thatthe subject has Alzheimer's disease or the subject is at a high risk ofdeveloping the disease if the mtDNA from said subject presents thenucleotide C at the polymorphic position 16519 according to the sequencedefined under accession number NC_012920 in 74% of mtDNA molecules. In apreferred embodiment of the invention it is considered that the subjecthas Alzheimer's disease or that the subject is at a high risk ofdeveloping said disease if the MtDNA of said subject presents thenucleotide C at the polymorphic position 16519 according to the sequencedefined under the accession number NC_012920 in 78% of mtDNA molecules.

As the person skilled in the art knows, a sample containingmitochondrial DNA can be homoplasmic or heteroplasmic. The term“heteroplasmy” or “heteroplasmic mitochondrial DNA” as used herein,refers to mitochondrial DNA of a subject that consists of a mixture ofDNA from at least two different genotypes of mitochondria. The term“homoplasmia” or “homoplasmic mitochondrial DNA” as used herein refersto mitochondrial DNA of a subject that is formed by a single genotype ofDNA from a single mitochondrial genotype.

In a specific embodiment, in the context of the present invention thesample of mitochondrial DNA is homoplasmic, in which case allmitochondria present in the subject contains identical genetic materialso all mitochondria of a subject present in their genome nucleotide C atthe polymorphic position 16519 according to the sequence defined underthe accession number NC_012920. In those embodiments wherein the sampleof mitochondrial DNA is homoplasmic, the fourth method of the inventionallows to diagnose or determine the risk of developing Alzheimer'sdisease in a subject by detecting the C nucleotide in the polymorphicposition 16519 according to the sequence defined under accession numberNC_012920 in said mitochondrial DNA sample; that is, the detection ofthe nucleotide C at the polymorphic position 16519 according to sequencedefined under the accession number NC_012920 in a sample of homoplasmicmitochondrial DNA from a subject is indicative that the subject suffersfrom Alzheimer's disease or that the subject has an elevated risk ofdeveloping the disease. Conversely, the detection of the nucleotide T atthe polymorphic position 16519 according to the sequence as definedunder the accession number NC_012920 in a sample of homoplasmicmitochondrial DNA, indicates that the subject is not suffering fromAlzheimer's disease or that the subject has a low risk of developing thedisease.

In another specific and preferred embodiment of the invention, themitochondrial DNA sample is heteroplasmic, i.e. mtDNA subject is fromtwo mitochondrial populations whose genetic material are not identicalto each other. According to this invention, heteroplasmy refers to thefact that a percentage of said mitochondria of said subject present inits genome the oligonucleotide C at the polymorphic position 16519according to the sequence defined under accession number NC_012920 andthe remaining percentage of mitochondria of said subject presents theoligonucleotide T in its in its genome at the polymorphic position 16519according to the sequence defined under accession number NC_012920.

The person skilled in the art will understand that the identification ofthe presence of polymorphism in at least 60% of mtDNA molecules isequally useful in order to select a subject for submission to preventivetreatment of Alzheimer's disease, in cases where the mtDNA of thesubject present heteroplamsy, as in the cases where the mtDNA of thesubject present homoplasmy, the totality of molecules will present thenucleotide T or the nucleotide C at position 16519, in which case thepercentage of mtDNA molecules with polymorphisms indicative that thepatient is a candidate to receive preventative treatment for Alzheimer'sdisease is either 100% or 0%. Like that, in the case of subjects whosemtDNA is heteroplasmic, there will be a population of MtDNA moleculesthat present the T nucleotide in position 16519 and a second populationof molecules that present the C nucleotide in position 16519. In thatcase, it is considered that the patient is candidate for a preventivetreatment of Alzheimer's disease when the percentage of mtDNA moleculesthat present nucleotide C at position 16519 is equal to or greater than60%.

The determination of homoplasmy and heteroplasmy and the percentage ofheteroplasmy or the “degree of heteroplasmy” can be determined by anytechnique known by a person skilled in the art non-limiting illustrativeexamples of techniques to allow to determine whether a sample ofmitochondrial DNA is heteroplastic include but are not limited tosouthern-blot, or PCR-RFLP of mtDNA sequencing. Briefly, the PCR-RFLPtechnique is based on the fact that normally the presence of a SNP in asample is associated with the creation or destruction of specificsequences or target of one or more restriction enzymes. Heteroplasmydetection by PCR-RFLP technique is a first stage in the amplification ofthe genetic material region containing the polymorphism to be detected,by using specific oligonucleotides, followed by a second step where theamplified fragments are subjected to enzymatic digestion reaction in thepresence of appropriate restriction enzyme. Since the presence orabsence of the polymorphism in the sample is associated with thepresence or absence of target specific restriction pattern obtainedfragment sizes determine whether the sample is formed by a uniquepattern of bands, in which case the sample is homoplasmic. If, however,the analysis determines the presence of two banding patterns,corresponding to two different mitochondrial DNA populations, then themtDNA sample is heteroplasmic.

The term “single nucleotide polymorphism” or “single nucleotidepolymorphism” or “SNP”, as used herein, refers to a variation in thenucleotide sequence of a nucleic acid that occurs in a single nucleotide(A, C, T or G), where every possible sequence is present in a proportionequal to or greater than 1% of the population. These polymorphismsappear when a single nucleotide in the genome is altered (for example bysubstitution, addition or deletion). Each version of the sequence withrespect to the polymorphic site is referred to as an allele of thepolymorphic site. SNPs tend to be evolutionarily stable from generationto generation and, as such, can be used to study specific geneticabnormalities in a population.

The polymorphic variant of the invention is the position 16519 based onthe numbering defined by the number NC_012920 in the NCBI database. Thepolymorphic variant contains a C in said position.

The terms “sequence determination of a SNP” or “detecting a SNP” areused interchangeably herein, and refer to the determination of asequence of a particular SNP in the subject under study. Determining thesequence of the SNP can be performed by various processes known to theperson skilled in the art.

In some specific embodiments of the invention, the sample comprisingmitochondrial DNA is selected from a biopsy of a solid tissue orbiofluid. The samples can be obtained by conventional methods known tothe person skilled in the art.

In an even more specific embodiment, the biofluid is selected fromperipheral blood or cerebrospinal fluid.

In an even more specific embodiment, said solid tissue is brain tissue.

If the material in which it is desired to determine said SNP accordingthe present method is a solid tissue or biofluid, preferably there is aprior nucleic acid extraction from the sample using any suitabletechnique for this.

In a preferred embodiment of the invention, the DNA fraction suitablefor the implementation of the invention is the total DNA. DNA extractionmay be carried out using any method known to the person skilled in theart as has been detailed in the first method of the invention.

If desired, the present method can be carried out in samples whichpreviously the mitochondrial fraction has been isolated and subsequentlythe DNA thereof has been isolated. The isolation of the mitochondrialfraction can be performed using any known method of cell fractionationand have been detailed in the first method of the present invention.

After isolating and amplifying (if necessary) the nucleic acid, the SNPsequence of the invention is detected by any method or technique capableof determining nucleotides present in a SNP or polymorphism. Forexample, a SNP can be detected by performing sequencing,mini-sequencing, hybridization, restriction fragment analysis,oligonucleotide ligation assay, allele-specific PCR, or a combinationthereof. As such, the systems and methods limited to, nucleic acidsequencing, hybridization methods and array technology (e.g. technologyavailable BioSciences Aclara, Affymetrix, Agilent Technologies, Inc.Illumina, etc.); can also be used in techniques based on mobility shiftof nucleic acid fragments amplified, for example Single StrandedConformational Polymorphism (SSCP), denaturing gradient gelelectrophoresis (DGGE), Chemical Mismatch Cleavage (CMC), RestrictionFragment Polymorphisms (RFLP), PCR-RFLP, WAVE analysis and the like(Methods Mol. Med. 2004; 108: 173-88). Of course, this list is merelyillustrative and in no way limiting. The experts in the field may useany method appropriate to achieve such detection.

In another specific embodiment, determining the sequence of said SNP isperformed by PCR-RFLP.

In a specific embodiment, the fourth method of the invention correspondsto an in vitro method to diagnose early stages of AD. The term “earlystages of Alzheimer's disease” as used herein, refers to Alzheimer'sdisease stage I-II according to Braak scale defined in the context ofthe first method of the invention.

Fifth Method of the Invention

In the fifth feature, the invention corresponds to an in vitro method toselect a subject for submission to a preventive treatment of Alzheimer'sdisease that involves determining in a sample containing mitochondrialDNA from said subject, the nucleotide at the polymorphic position 16519according to the sequence with accession number NC_012920 in the NCBIdatabase, wherein the detection of the C nucleotide at said polymorphicposition or the presence of the nucleotide C at said polymorphicposition in at least 60% of the mitochondrial DNA molecules indicativethat the subject is a candidate to receive treatment aimed at preventingAlzheimer's disease.

The terms “Alzheimer”, “treatment”, “sample”, “mitochondrial DNA” and“polymorphism” as well as methods for obtaining the sample and detectinga polymorphism have been detailed in the context of the first, second,third and fourth method of the invention and are used here with the samemeaning.

According to the fifth method of the invention it is considered that thesubject is a candidate to be submitted to treatment aimed at preventingAlzheimer's disease if the mtDNA of the subject has nucleotide C at thepolymorphic position 16519 according to the sequence defined underaccess number NC_012920 when the percentage of mtDNA molecules that havesaid nucleotide in said position is at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% or more. Ina specific embodiment, it is considered that the subject is eligible toreceive preventive treatment of Alzheimer's disease if the mtDNA of saidsubject presents the nucleotide C at the polymorphic position 16,519according the sequence defined under accession number NC_012920 in 60%of mtDNA molecules.

The person skilled in the art will understand that the identification ofthe presence of the polymorphism in at least 60% of mtDNA molecules isequally useful to select a subject to be submitted to preventivetreatment of Alzheimer's disease in cases where mtDNA of the subjectpresent heteroplasmic as in the cases which it presents homoplasmic.Like that, as in the case of subjects whose mtDNA is homoplasmic, thetotality of the molecules will present the nucleotide T or thenucleotide C at position 16519, in which case the percentage of mtDNAmolecules with the polymorphisms indicative that the patient is acandidate to receive preventative treatment for Alzheimer's disease iseither 100% or 0%. Like that, in the case of subjects whose mtDNA isheteroplasmic, there will be a population of mtDNA molecules whichpresent the T nucleotide at position 16519 and a second population ofmolecules will present the C nucleotide at the position 16519. In thatcase, it will be considered that the patient is a candidate forpreventive treatment of Alzheimer's disease when the percentage ofmolecules of mtDNA present the nucleotide C in position 16519 is lessthan or greater than 60%.

In some specific embodiments of the invention, the sample comprisingmitochondrial DNA is selected from a biopsy of a solid tissue orbiofluid, samples can be obtained by conventional methods known to theperson skilled in the art.

In a specific embodiment, in the context of the present invention thesample of mitochondrial DNA is homoplasmic, in which case allmitochondria present in the subject contains identical genetic materialso all mitochondria of a subject present nucleotide C in their genome inthe polymorphic position 16519 according to the sequence under theaccession number NC_012920. In those embodiments where the mitochondrialDNA sample is homoplasmic, the fifth method of the invention allowsselecting a patient to be submitted to preventive treatment ofAlzheimer's disease through the detection of the nucleotide C at thepolymorphic position 16519 according to the sequence defined underaccession number NC_012920 in said mitochondrial DNA sample; i.e.detection of nucleotide C at the polymorphic position 16519 according tothe sequence defined under accession number NC_012920 in a sample ofhomoplasmic mitochondrial DNA from a patient is indicative that thesubject is eligible to receive preventive treatment of Alzhemier'sdisease. Conversely, detection of nucleotide T in at polymorphicposition 16519 according to the sequence defined under accession numberNC_012920 in a sample of homoplasmic mitochondrial DNA from a patient isindicative that said patient is not a candidate to receive preventivetreatment for Alzheimer's disease.

In another specific and preferred embodiment of the invention, themitochondrial DNA sample is heteroplasmic, i.e. the mtDNA of the subjectis of two mitochondrial populations whose genetic material are notidentical to each other. According to the present invention heteroplasmyrefers to a percentage of mitochondria of said subject that present inits genome the oligonucleotide C in the polymorphic position 16519according to the sequence defined under the accession number NC_012920and the remaining percentage of mitochondria of said subject presentoligonucleotide T in the genome in the polymorphic position 16519according to the sequence defined under accession number NC_012920.

Methods for determining whether a sample is homoplasmic or heteroplasmicas well as to determine the degree of heteroplasmy of a sample have beendetailed in the context of the fourth method of the invention and areincorporated herein by reference.

In an even more specific embodiment, the biofluid is selected fromperipheral blood or cerebrospinal fluid.

In an even more specific embodiment, said solid tissue is brain tissue.

If the material in which it is desired to determine said SNP accordingthe present method is a solid tissue or biofluid, preferably there is aprior nucleic acid extraction from the sample using any suitabletechnique for this. In a preferred embodiment of the invention, the DNAfraction suitable for the implementation of the invention is the totalDNA. The extraction of DNA may be carried out using any appropriatemethod known to persons skilled in the art as has been detailed in thefirst method of the invention.

In another specific embodiment, determining the sequence of said SNP isperformed by PCR-RFLP.

Polynucleotides of the Invention

In another feature, the present invention corresponds to a nucleic acid(hereinafter “first polynucleotide of the invention”) comprising atleast 9 contiguous polynucleotides in a mtDNA region wherein said regioncomprises at least one methylation site selected from the CpG sites inthe D-loop region shown in Table 1.

The term “polynucleotide” as used herein, refers to DNA or RNA moleculesof more than 13 bases in length. The polynucleotides of the inventionare preferably DNA molecules of at least 14, at least 15, at least 16,at least 18, at least 20, at least 25, at least 30, at least 35, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100 or more bases in length.

In a specific embodiment, the first polynucleotide of the inventioncomprises at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 20, at least 25, at least 30 ormore contiguous nucleotides to at least one CpG site selected from Table1.

In another feature, the present invention relates to a nucleic acid(hereinafter “second polynucleotide of the invention”) comprising atleast 9 contiguous polynucleotide in a mitochondrial DNA region whereinsaid region contains at least one methylation site selected from CpGsites in the ND1 gene shown in Table 2

In a specific embodiment, the second polynucleotide of the inventioncomprises at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 20, at least 25, at least 30 ormore contiguous nucleotides, to said at least one CpG site selected fromTable 2. In another feature, the present invention relates to a nucleicacid (hereinafter “second polynucleotide of the invention”) comprisingat least 9 contiguous polynucleotide in a mitochondrial DNA regionwherein said region comprises at least one methylation site selectedfrom the CHG sites in the D-loop region shown in Table 3.

In a specific embodiment, the third polynucleotide of the inventioncomprises at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 20, at least 25, at least 30 ormore contiguous nucleotides, to said at least one CHG site selected fromTable 3.

In another feature, the present invention relates to a nucleic acid(hereinafter “fourth polynucleotide of the invention”) comprising atleast 9 contiguous polynucleotide in a mitochondrial DNA region whereinsaid region contains at least one methylation site selected from CHGsites of the ND1 gene shown in Table 4.

In a specific embodiment, the fourth polynucleotide of the inventioncomprises at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 20, at least 25, at least 30 ormore contiguous nucleotides to said at least one CHG site selected fromTable 4.

In another aspect, the present invention refers to a nucleic acid(hereinafter 35 hereinafter “fifth polynucleotide of the invention”)comprising at least 9 contiguous polynucleotide in a mtDNA regionwherein said region comprises at least one methylation site selectedfrom the CHH sites in the D-loop region shown in Table 5.

In a specific embodiment, the fifth polynucleotide of the inventioncomprises at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 20, at least 25, at least 30 ormore contiguous nucleotides, to said at least one CHH site selected fromTable 5.

In another feature, the present invention corresponds to a nucleic acid(hereinafter “sixth polynucleotide of the invention”) Comprising atleast 9 contiguous polynucleotide in a mitochondrial DNA region whereinsaid region comprises at least one methylation site selected from theCpG sites in the D-loop region shown in Table 1, wherein thecorresponding position of cytosine in said CpG site is uracil.

In a particular embodiment, the sixth polynucleotide of the inventioncomprises at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 20, at least 25, at least 30 ormore contiguous nucleotides, to said at least one CpG site selected fromTable 1 wherein the corresponding position of cytosine in said CpG siteis uracil.

In another aspect, the present invention refers to a nucleic acid(hereinafter “seventh polynucleotide of the invention”) comprising atleast 9 contiguous polynucleotides in a mitochondrial DNA region whereinsaid region comprises at least one methylation site selected from theCpG sites of the ND1 gene shown in Table 2, wherein the correspondingposition of cytosine in said CpG site is uracil.

In a particular embodiment, the seventh polynucleotide of the inventioncomprises at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 20, at least 25, at least 30 ormore contiguous nucleotides to said at least one CpG site selected fromTable 2 wherein the corresponding position of cytosine in said CpG siteis uracil.

In another feature, the present invention refers to a nucleic acid(hereinafter “eight polynucleotide of the invention”) Comprising atleast 9 contiguous polynucleotides in mitochondrial DNA region whereinsaid region comprises at least one methylation site selected from theCHG sites in the D-loop region shown in Table 3 wherein thecorresponding position of cytosine in said CHG site is uracil.

In a particular embodiment, the eight polynucleotide of the inventioncomprises at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 20, at least 25, at least 30 ormore contiguous nucleotides to said at least one CHG site selected fromTable 3, wherein the corresponding position of cytosine in said CHG siteis uracil.

In another feature, the present invention relates to a nucleic acid(hereinafter “ninth polynucleotide of the invention”) comprising atleast 9 contiguous polynucleotides in a mitochondrial DNA region whereinsaid region comprises at least one methylation site selected from theCHG sites of the ND1 gene shown in Table 4, wherein the correspondingposition of cytosine in said CHG site is uracil.

In a specific embodiment, the ninth polynucleotide of the inventioncomprises at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 20, at least 25, at least 30 ormore contiguous nucleotides, that is said, at least one CHG siteselected from Table 4, wherein the corresponding position of cytosine insaid CHG site is uracil.

In another feature, the present invention relates to a nucleic acid(hereinafter “tenth polynucleotide of the invention”) Comprising atleast 9 contiguous polynucleotide in a mitochondrial DNA region whereinsaid region comprises at least one methylation site selected from theCHH sites in the D-loop region shown in Table 5, wherein thecorresponding position of cytosine in said CHH site is uracil.

In a specific embodiment, the tenth polynucleotide of the inventioncomprises at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 20, at least 25, at least 30 ormore contiguous nucleotides to said at least one CHH site selected fromTable 5, wherein the corresponding position of cytosine in said CHH siteis uracil.

In another feature, the invention relates to a polynucleotide thatspecifically hybridises with said first, second, third, fourth, fifth,sixth, seventh, eighth, ninth and tenth polynucleotides of theinvention.

The expression “that specifically hybridises” or “capable of hybridisingin a specific form”, as used herein, refers to the ability of anoligonucleotide or of a polynucleotide of specifically recognizing asequence of a CpG, CHG or CHH site. As used herein the term“hybridization” It is the process of combining two nucleic acidmolecules or single-stranded molecules with a high degree of similarityresulting in a simple double-stranded molecule by specific pairingbetween complementary bases. Normally hybridization occurs under verystringent conditions or moderately stringent conditions.

As known in the technology, the “similitude” between two nucleic acidmolecules is determined by comparing the nucleotide sequence of amolecule to the nucleotide sequence of a second molecule. The variantsaccording to the present invention include nucleotide sequences that areat least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar or identicalto the sequence of said at least one CpG, CHH and CGH site selected fromthe sites shown in Tables 1 to 5. The degree of identity between twonucleic acid molecules is determined using computer algorithms andmethods that are widely known to persons skilled in the art. Theidentity between two amino acid sequences preferably determined byBLASTN algorithm (BLAST Manual, Altschul et al, 1990, NCBI NLM NIHBethesda, Md. 20894, Altschul, S., et al, J. Mol Biol. 215: 403-10).

The “rigor” of hybridization reactions is readily determined by anordinary expert in the field, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridisable sequence, the higher the relativetemperature that can be used. As a result, it is deduced that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

The term “stringent conditions” or “high stringency conditions” as usedherein, typically: (1) employ low ionic strength and high temperaturefor washing, for example 0.015 M sodium chloride/0.0015 citrate/0.1%sodium dodecyl sulfate at 50° C. sodium; (2) employ during hybridizationa denaturing agent such as formamide, for example, 50% (v/v) formamidewith 0.1% bovine serum albumin/0.1% FicoII/0.1% polyvinylpyrrolidone/50mM buffer sodium phosphate at pH 6.5 with 750 mM sodium chloride, 75 mMcitrate sodium at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 MNaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5×Denhardt's solution, DNA sonicated salmon sperm(50 mg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide,followed by a high-stringency wash 0.1×SSC consisting containing EDTA at55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al, Molecular Cloning. A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic force and % SDS) lessstringent than those described above. An example of moderately stringentconditions is overnight incubation at 37° C. in a solution comprising:20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mMsodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate and20 mg/ml salmon sperm DNA denatured fragmented, followed by washing thefilters in 1×SSC at about 35 37-50° C. the person skilled in the artwill know how to adjust the temperature, ionic strength, etc. ifnecessary to accommodate factors such as probe length and the like.

Kits of the Invention

In another feature, the present invention relates to a kit (hereinafter“first kit of the invention”) Comprising at least one oligonucleotidecapable of specifically hybridizing and methylation-dependent mannerwith a mitochondrial DNA sequence comprising a methylation site selectedfrom the group formed by:

-   -   (i) the CpG sites in the D-loop region shown in Table 1,    -   (ii) the CpG sites in the ND1 gene shown in Table 2,    -   (iii) the CHG sites in the D-loop region shown in Table 3,    -   (iv) the CHG sites in the ND1 gene shown in Table 4, and/or    -   (v) the CHH sites in the D-loop region shown in Table 5

The terms “CpG site”, “CHG site”, “CHH site”, “D-loop region” and “ND1gene” have been described in detail in the context of the first methodof the invention and the term “capable of hybridising in a specificform” is defined in the context of the polynucleotides of the invention.These terms are used with the same meaning in the context of theinvention kits.

In a preferred embodiment, the oligonucleotides are part of theinvention kit which is capable of specifically hybridizing dependentlyfrom methylation with a mitochondrial DNA sequence comprising amethylation site selected from the group consisting of:

-   -   (i) the CpG sites in the D-loop region shown in Table 1,    -   (ii) the CpG sites in the ND1 gene shown in Table 2,    -   (iii) the CHG sites in the D-loop region shown in Table 3,    -   (iv) the CHG sites in the ND1 gene shown in Table 4, and/or    -   (v) the CHH sites in the D-loop region shown in Table 5

They constitute at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90% orat least 100% of the total amount of the oligonucleotides that form thekit. In further embodiments, said oligonucleotides are at least 55% atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of the total amount of oligonucleotides which form the kit.

Suitable kits include various reagents for use according to the presentinvention, in suitable containers and packaging materials, includingtubes, vials, and shrink wrap packages and blow molders. In addition,invention kits can contain instructions for the simultaneous, sequentialor separate use of the various components found in the kit. Suchinstructions may be in the form of printed material or in the form ofelectronic media capable of storing instructions so that they can beread by a subject, such as electronic storage media (magnetic disks,tapes and the like), optical media (CD-ROM, DVD) and the like.Additionally, or alternatively, the media can contain Internet addressesthat provide such instructions. Suitable materials for inclusion in anexemplary kit according to the present invention comprise one or more ofthe following: reagents capable of amplifying a specific sequence of adomain either total or mtDNA DNA without the need to carry out PCR;reagents required to discriminate between the different possible allelesin the sequence domains amplified by PCR amplification or non-PCR (e.g.,restriction endonuclease, hybridizing oligonucleotides preferably CpG,CHG and/or CHH sites methylated or unmethylated, including thosemodified to contain enzymes or fluorescent chemical groups that amplifythe oligonucleotide signal and make that the discrimination between CpG,CHG and/or CHH methylated or unmethylated sites CHH is more robust); orreagents required for physically separating the various productsamplified regions (e.g., agarose or polyacrylamide and a buffer for usein electrophoresis, HPLC columns, SSCP gels, formamide gels or a supportmatrix for MALDI-TOF).

The term “oligonucleotide” as used herein, refers to a DNA molecule orshort RNA, with up to bases in length. Oligonucleotides of the inventionare preferably DNA molecules at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 15, at least 20, at least 30, at least 40, at least 45 or 50 basesof length.

As used in the kit of the invention, at least one oligonucleotidecapable of hybridizing with at least a sequence of a CpG site in theD-loop region selected from the CpG sites shown in Table 1, at least oneoligonucleotide capable of hybridizing with at least one sequence of aCpG site in the ND1 gene shown in Table 2, at least one oligonucleotidecapable of hybridizing with at least one sequence of a CHG site in theD-loop region shown in Table 3, at least one oligonucleotide capable ofhybridizing with at least one sequence of a CHG site in the ND1 geneshown in Table 4 and/or at least one oligonucleotide capable ofhybridizing with at least one sequence of a CHH site in the D-loopregion shown in Table 5, in a methylation specific manner, it is used asa primer to amplify the region containing said CpG, CHG and/or CHHsite(s) Alternatively, at least one oligonucleotide can also be used asa probe to detect such CpG, CHG or CHH methylated or unmethylated sites.

In a preferred embodiment of the first kit of the invention compriseoligonucleotides capable of specifically hybridizing with all CpG sitesin the D-loop region shown in Table 1, all CpG sites of the ND1 geneshown in Table 2, with all of the CHG sites of the D-loop region shownin Table 3, with all of CHG sites in the ND1 gene shown in Table 4and/or all CHH sites in the D-loop region shown in Table 5.

If desired, the first kit of the invention may comprise a firstoligonucleotide capable of specifically hybridizing withbisulfites-treated oligonucleotide comprising at least one sequence of aCpG site in the D-loop region shown in Table 1 when said CpG site ismethylated, and at least one oligonucleotide or polynucleotide capableof specifically hybridizing with the same bisulfite-treatedoligonucleotide comprising at least one sequence of a CpG site in theD-loop region when said CpG site is unmethylated; and/or the kit maycomprise a first oligonucleotide capable of specifically hybridizingwith bisulfites-treated oligonucleotide comprising at least one sequenceof a CpG site in the ND1 gene shown in Table 2 when said CpG site ismethylated, and at least one oligonucleotide or polynucleotide capableof specifically hybridizing with the same bisulfite-treatedoligonucleotide comprising at least one sequence of a CpG site of theD-loop region when said CpG site is unmethylated; and/or the kit maycomprise a first oligonucleotide capable of specifically hybridizingwith a bisulfite-treated oligonucleotide comprising at least onesequence of a CHG site in the D-loop region shown in Table 3 when saidCHG site is methylated, and at least one oligonucleotide orpolynucleotide capable of specifically hybridizing with the samebisulfite-treated oligonucleotide that comprises at least a sequence ofa CHG site of the D-loop region when said CHG site is not methylated;and/or the kit may comprise a first oligonucleotide capable ofspecifically hybridizing with bisulfite-treated oligonucleotidecomprising at least one sequence of a CHG site in the ND1 gene shown inTable 4 when said site CHG is methylated, and at least oneoligonucleotide or polynucleotide capable of hybridizing specificallywith the same bisulfites-treated oligonucleotide that contains at leastone sequence of a CHG site in the D-loop region when said CHG site isunmethylated; and/or the kit may comprise a first oligonucleotidecapable of specifically hybridizing with bisulfites-treatedoligonucleotide comprising at least a sequence of a CHH site in theD-loop region shown in Table 5 when said CHH site is methylated and atleast one oligonucleotide or polynucleotide capable of specificallyhybridizing the same bisulfite-treated oligonucleotide comprising atleast one sequence of a CHH site in the D-loop region when said CHH siteis unmethylated.

For hybridization of an unmethylated CpG site, specific primers thathybridize with non-methylated DNA, have, preferably, a T in the CG parin “to distinguish it from the C retained in methylated DNA. It ispreferable that primers contain relatively few Cs or Gs in the sequencesince the Cs will be absent in the sense primer and the Gs absent in theantisense primer (cytosine converted into uracil, which is amplified asthymidine in the amplification product). Accordingly, for hybridizationto a methylated CpG site, primers that specifically hybridize methylatedDNA preferably they have a C at the 3′CG pair.

In another feature, the invention relates to a kit (hereinafter “secondkit invention”) comprising at least one oligonucleotide capable ofhybridizing.

Specifically, with a zone in 5′ to 3′ position with respect to amethylation site of mitochondrial DNA selected from the group consistingof:

-   -   i. the CpG sites in the D-loop region shown in Table 1,    -   ii. the CpG sites in the ND1 gene shown in Table 2,    -   iii. the CHG sites in the D-loop region shown in Table 3,    -   iv. the CHG sites in the ND1 gene shown in Table 4, and/or    -   v. the CHH sites in the D-loop region shown in Table 5        where methylated cytosine in said position has converted to        uracil or another base that is distinguishable from cytosine in        its hybridization properties.

In a preferred embodiment, the oligonucleotides that form part of thekit of the invention and which are capable of specifically hybridizingto a region or position 5′ or position 3′ with respect to a methylationsite in the mitochondrial DNA selected from the group formed by:

-   -   i. the CpG sites in the D-loop region shown in Table 1,    -   ii. the CpG sites in the ND1 gene shown in Table 2,    -   iii. the CHG sites in the D-loop region shown in Table 3,    -   iv. the CHG sites in the ND1 gene shown in Table 4, and/or    -   v. the CHH sites in the D-loop region shown in Table 5

They constitute at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90% orat least 100% of the total amount of the oligonucleotides that form thekit. In further embodiments, said oligonucleotides constitute at least55% at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% of the total amount of oligonucleotides that make upthe kit.

In a specific embodiment, the second kit of the invention furthercomprises one or more reagents to convert unmethylated cytosine touracil or another base that is differentially detectable to cytosine interms of hybridization properties.

In a preferred embodiment, the one or more reagents to convertunmethylated cytosine to uracil or another base that is detectablydifferent from cytosine in terms of hybridization properties isbisulfite, preferably sodium bisulfite. The reagent capable ofconverting unmethylated cytosine to uracil or another base that isdifferentially detectable to cytosine in terms of hybridizationproperties is metabisulfite, preferably sodium metabisulfite.

The term “conversion reagent” and its details are described in detail incontext of the diagnostic method of the invention and used with the samemeaning in the context of the kit according to the invention.

In a specific embodiment, the second kit of the invention comprises atleast one oligonucleotide comprising a sequence selected from thesequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and/or SEQID NO: 5.

In another feature, the invention refers to the use of the first and/orsecond kit of the invention to determine the pattern of mitochondrialDNA in a subject or to determine the diagnosis of a neurodegenerativedisease in a subject selected from Alzheimer's disease and Parkinson'sdisease.

The invention is described by the following examples which are to beconstrued as merely illustrative and not limitative of the scope of theinvention

EXAMPLES

Materials and Methods

Study Design and Subjects Included

The study was conducted on 44 samples, including pathology related toAlzheimer's disease (AD), Parkinson's disease (PD) and control cases.The samples were processed in a plate divided into two lanes. In lane 1amplicons D-Loop ND1 and samples were analyzed in entorhinal cortexcases related pathology EA and corresponding controls (Table 7). In lane2 D-Loop amplicons were analyzed on samples of the substantia nigra inPD and its corresponding controls (Table 7). Each patient was identifiedin the primers used with MID (multiplex Identifier) (Tables 7 and 8).Methylation in CpG sites and non CpG sites (CHG and CHH, where H=A, T,or C) was analyzed using a pyrosequencer 454 GS FLX Titanium Roche thatgenerated 569.684 sequences in lane 1, whose lengths varied from 40-1098base pairs (bp) with an average length of about 417 bp. In lane 2 thenumber of sequences obtained was 513.579, whose length ranged from 40 to933 bp (466 bp average length). Alignments of the sequences obtained foreach MID, amplicon and lane against their respective reference sequenceswere noted and the percentages of identity between these were nearly100%. The mean and median rates of bisulphite conversion for each locusand MID were analyzed.

The number of unmethylated sequences exceeded the number of methylatedsequences identified in each site and some methylation sites were notpresent. Those sequences after alignment were found to have, at least,one not present site of the methylation pattern not, were removed fromthe analysis to avoid any bias at the time of quantization. Thiscircumvents approximation analysis of putative mitochondrial pseudogeneswhose amplicons presented nearly 100% of identity with mitochondrial DNAwhen analyzed in NCBI BLAST. Most CpG, CHG, and CHH sites analyzed wereunmethylated. However, different methylation sites could be identified.

TABLE 6 Number of differentially methylated sites. FRD: p-value adjustedBenjamini method and Hocheberg (1995) AD I-II No. C vs. C vs. vs. C ofAD AD AD vs. Lane Amplicon Site Criteria Sites I-II III-IV III-IV PD L1D-loop CG FDR <0.01 18 17 14 12 — L1 D-loop CG FDR <0.05 18 17 15 16 —L1 ND1 CG FDR <0.01 13 7 7 0 — L1 ND1 CG FDR <0.05 13 7 7 0 — L1 D-loopCHG FDR <0.01 16 13 10 1 — L1 D-loop CHG FDR <0.05 16 14 12 7 — L1 ND1CHG FDR <0.01 9 6 5 0 — L1 ND1 CHG FDR <0.05 9 7 5 0 — L1 D-loop CHH FDR<0.01 52 0 0 34 — L1 D-loop CHH FDR <0.05 52 23 0 43 — L1 ND1 CHH FDR<0.01 72 0 0 0 — L1 ND1 CHH FDR <0.05 72 0 0 0 — L2 D-loop CG FDR <0.0118 — — — 17 L2 D-loop CG FDR <0.05 18 — — — 17 L2 D-loop CHG FDR <0.0116 — — — 14 L2 D-loop CHG FDR <0.05 16 — — — 14 L2 D-loop CHH FDR <0.0152 — — — 44 L2 D-loop CHH FDR <0.05 52 — — — 44

TABLE 7 Summary of key clinical features and neuropathological humancases analyzed. Break stages Alzheimer's disease (AD) indicate thedegree of presence of neurons with fibrillary neurodegeneration (romannumerals) and senile plaques (letters) following the classification ofBreak. Break stages for Parkinson's disease (PD) refers to the degree ofpresence of the protein α-synuclein (Lewy bodies). Stage of Stage ofTime Region of Break for Break for postmortem MID Lane brain DiagnosisAD PD Gender Age (h) 1 L1 entorhinal CONTROL 0/0 0 M 56 5 2 L1entorhinal CONTROL 0/0 0 M 56 3.45 3 L1 entorhinal CONTROL 0/0 0 F 558.3 4 L1 entorhinal CONTROL 0/0 0 F 66 4.15 5 L1 entorhinal CONTROL 0/00 F 64 5 6 L1 entorhinal CONTROL 0/0 0 F 52 5.45 7 L1 entorhinal CONTROL0/0 0 M 57 5.20 8 L1 entorhinal CONTROL 0/0 0 M 64 3.3 9 L1 entorhinalAD I/A 0 F 57 5 associated 10 L1 entorhinal AD I/A 0 F 64 2.15associated 11 L1 entorhinal AD I 0 M 59 16.3 associated 12 L1 entorhinalAD II/A 0 F 86 4.15 associated 13 L1 entorhinal AD I/A 0 M 67 14.4associated 14 L1 entorhinal AD II/A 0 M 66 4.55 associated 15 L1entorhinal AD I/A 0 F 63 8.05 associated 16 L1 entorhinal AD II/A 0 F 765.45 associated 17 L1 entorhinal AD III/A 0 F 71 6.45 associated 18 L1entorhinal AD III-A 0 F 77 11.5 associated 19 L1 entorhinal AD III/B 0 M86 3.1 associated 20 L1 entorhinal AD IV/C 0 F 69 8.1 associated 9 L1entorhinal AD I/A 0 F 57 5 associated 10 L1 entorhinal AD I/A 0 F 642.15 associated 11 L1 entorhinal AD I 0 M 59 16.3 associated 12 L1entorhinal AD II/A 0 F 86 4.15 associated 21 L1 entorhinal AD III 0 F 793.4 associated 22 L1 entorhinal AD IV/C 0 M 75 6.1 associated 23 L1entorhinal AD III/A 0 F 74 4 associated 24 L1 entorhinal AD III/0 0 M 873.3 associated 1 L2 SN CONTROL 0/0 0 F 78 3.4 2 L2 SN CONTROL 0/0 0 M 663 3 L2 SN CONTROL 0/0 0 F 71 8.3 4 L2 SN CONTROL 0/0 0 M 30 4.1 5 L2 SNCONTROL 0/0 0 M 47 5 6 L2 SN CONTROL 0/0 0 M 67 5 7 L2 SN CONTROL 0/0 0M 39 9.15 8 L2 SN CONTROL 0/0 0 M 85 5.45 9 L2 SN CONTROL 0/0 0 F 469.35 10 L2 SN CONTROL 0/0 0 F 77 3.15 11 L2 SN PD 0/0 3.4 M 68 9.2 12 L2SN PD 0/0 3.4 F 81 6.3 13 L2 SN PD 0/0 3.4 M 76 12.3 14 L2 SN PD 0/0 3.4F 77 3.3 15 L2 SN PD 0/0 3.4 F 78 27.3 16 L2 SN PD 0/0 3.4 F 69 4.3 5 L2SN CONTROL 0/0 0 M 47 5 6 L2 SN CONTROL 0/0 0 M 67 5 7 L2 SN CONTROL 0/00 M 39 9.15 8 L2 SN CONTROL 0/0 0 M 85 5.45 9 L2 SN CONTROL 0/0 0 F 469.35 10 L2 SN CONTROL 0/0 0 F 77 3.15 11 L2 SN PD 0/0 3.4 M 68 9.2 12 L2SN PD 0/0 3.4 F 81 6.3 13 L2 SN PD 0/0 3.4 M 76 12.3 14 L2 SN PD 0/0 3.4F 77 3.3 15 L2 SN PD 0/0 3.4 F 78 27.3 16 L2 SN PD 0/0 3.4 F 69 4.3 17L2 SN PD I/O 4 M 69 6 18 L2 SN PD 0/0 4 F 84 4.3 19 L2 SN PD 0/0 5 M 7612 20 L2 SN PD 0/0 5 M 80 7.3

TABLE 8 MID sequence associated with each case analyzed number MIDMID sequence SEQ ID NO:  1 ACGAGTGCGT  6  2 ACGCTCGACA  7  3 AGACGCACTC 8  4 AGCACTGTAG  9  5 ATCAGACACG 10  6 ATATCGCGAG 11  7 CGTGTCTCTA 12 8 CTCGCGTGTC 13  9 TAGTATCAGC 14 10 TCTCTATGCG 15 11 TGATACGTCT 16 12TACTGAGCTA 17 13 CATAGTAGTG 18 14 CGAGAGATAC 19 15 ATACGACGTA 20 16TCACGTACTA 21 17 CGTCTAGTAC 22 18 TCTACGTAGC 23 19 TGTACTACTC 24 20ACGACTACAG 25 21 CGTAGACTAG 26 22 TACGAGTATG 27 23 TACTCTCGTG 28 24TAGAGACGAG 29Human Brain Samples

Tissue samples were provided by the Neurological Tissue Bank, Universityof Barcelona—Hospital de Barcelona and the Bank of Institute ofNeuropathology, HUB-ICO-IDIBELL. The donation and procurement of sampleswas regulated by the ethics committee of both institutions. Half of eachbrain was maintained in buffered formalin 4% solution for morphologicaland histological study, while the other half was processed in coronalsections to be frozen at −80° C. to be available for biochemicalstudies. Neuropathological examination in all controls and pathologicalcases took place in thirty standardized sections of the brain,cerebellum and brain stem, which were stained with hematoxylin andeosin, and Klüver Barrera, or processed for immunohistochemistry forglial fibrillary acidic protein, microglial markers, beta-amyloid,phosphorylated tau (AT8 antibody) α-synuclein, β-crystallin, ubiquitinand TDP-43. Cases with pathology related to AD and PD were classifiedaccording to the current neuropathological criteria (Braak and Braak1991, 1999; Braak et al, 2003, 2006). Cases with mixed pathology(including vascular lesions) were excluded from this study. The brainsused as control belonged to individuals without neurologicalmanifestations and without injury in the neurological study. The medicalrecords were reexamined for each case, and cases with pathology relatedto AD (stages I-IV) were reevaluated through phone calls or interviewswith relatives, asking if they had evidence of any neurological orcognitive impairment. Only cases meeting these criteria were consideredin this work. All cases analyzed summarized in Table 7.

The average post-mortem interval samples entorhinal cortex was 4.98±1.57hours in controls, 7.51±5.13 hours in stages I-II, and 5.70±2.85 hoursstage III-IV; for samples of the substantia nigra intervals were5.59±2.46 hours and 9.23 in controls ±7.07 hours in the case of PD.

Murine Brain Samples

Transgenic mice APP/PS1 and wild strain were obtained from JacksonLaboratory (USA). The transgenic model expresses a murine/human chimeraAPP (Mo/HuAPP695swe: Swedish mutation APP) molecule and the humanvariant of presenilin 1 (PS1-DE9), both expression in neurons of thenervous system product. The animals were housed in standard conditionswith light and dark cycles of 12 hours, with unlimited access to foodand water. Stabling was conducted following the ethical guidelines(European Communities Council Directive 86/609/EEC) approved by thelocal ethics committee.

Total DNA Extraction

Total DNA was isolated human samples of the entorhinal cortex andsubstantia nigra (Table 7) using the DNeasy Blood and Tissue Kit(Qiagen, Madrid, Spain) following the manufacturer's instructions. TotalDNA samples murine was obtained from the frontal cortex using the sameprocedure.

Bisulfite Treatment

Three hundred nanograms of DNA were treated using bisulfite EZ DNA kitmethylation Kit (Zymo Research, Ecogen, Barcelona, Spain) following thesupplier's instructions. The bisulfite-treated DNA was resuspended in 30μl to reach a final concentration of 10 ng/ul. All samples were treatedwith bisulfite in parallel, using the same batch of reagent to avoiddifferences in the rate of bisulfite conversion between differentcommercial lots.

Design of the Primers for Amplicon FLX-Loop D and ND1

The primers for the FLX experiment were designed following theinstructions techniques for FLX sequencer Roche “Amplicon Fusion PrimerDesign Guidelines for GS FLX Titanium Series Lib-A Chemistry”. MeltingPrimers for amplicons containing a directional primer GS FLX Titaniumprimer A or primer B (including a key sequence (Key) Four bases) in Lot5-oligonucleotide premium, plus a specific sequence for mold at 3—firstfinal. Moreover, a MID sequence was added (multiplex Identifier) Betweenthe primer A (or primer B) and the specific sequence for subsequentautomated sample identification software after stepsgrouping/multiplexing and sequencing. The primers used contained thefollowing components: Primer forward (Primer A-Key-MID—specific sequencemold), 5′-CGTATCGCCTCCCTCGCGCCA (SEQ ID NO: 33)--MID TCAG—specifictemplate sequence 3′; Primer reverse (Primer B-Key-MID—specific templatesequence): 5′CTATGCGCCTTGCCAGCCCGC (SEQ ID NO: 34)--MID TCAG—specifictemplate sequence-3′. Specific template sequences for each of theamplicons: D-loop-direct, 5′-TAGGGGTTTTTTGATTATTATTTTT-3′(SEQ ID NO: 2)and D Loop-reverso, ACAAACATTCAATTATTATTATTATATCCT 5′-3′ (SEQ ID NO: 3);ND1-direct, 5′-ATGGTTAATTTTTTATTTTTTATTGTATTT-3′ (SEQ ID NO: 4) andND1-reverso, 5′-TAATTTAAATTTAATACTCACCCTAATCAA-3′(SEQ ID NO: 5). Theprimers used in this study were designed to avoid CpG sites. SequenceMIDs specific for each patient shown in Table 8. amplified regions(D-Loop: 16386-256; ND1: 3313-3686) are based on nucleotide position mapof human mtDNA (www.mitomap.org).

Preparation of Amplicon Library

The PCR amplicons for D-Loop and ND1 were performed according to themanual Amplicon Library Preparation Method Manual (GS FLX TitaniumSeries) Of Roche. PCRs for twenty nanograms total bisulfite-treated DNAwere used. DNA amplification bisulfite treated are carried out in areaction volume 25 ul. Each PCR reaction consisted of: 1× FastStart 10×Buffer #2 0.05 U/ul polymerase HiFi FastStart Polymerase (Roche), 200 nMof each dNTP, and 200 nM of each primer specific forward Y reverse. Theprimers were synthesized with a HPLC purification quality(Sigma—Aldrich, Madrid, Spain). Amplifications were performed in athermocycler Applied Biosystems Verity® (Applied Biosystems, Madrid,Spain) using the following conditions: 94° C. for 3 min and then 36cycles of 94° C. for 15 s, temperature annealing (61° C. ND1, and 62° C.D-Loop) for 45 s and 72° C. for 1 min, followed by a final extensionstep at 72° C. for 8 min and a final hold temperature at 4° C. twomicroliters of each PCR product were checked on an agarose gel stainedwith 1.5% SYBR® Safe DNA Gel Stain (Invitrogen, Madrid, Spain).

Purification of PCR

Purification of PCR products was performed using the kitAgencourt®AMPure®XP PCR Purification (Beckman Coulter, Madrid, Spain)following the instructions manual Roche Amplicon Library PreparationMethod Manual (GS FLX Titanium Series).

Quantification of Amplicons and Sequencing Libraries FLX

Quantification and quality control amplicon libraries and the rest ofFLX sequencing protocol was performed by the team of the Platform forGenomic Research Institute of Vall d'Hebron (VHIR, Barcelona, Spain).

Selection of Differentially Methylated Sites

Alignment and identification of CpG, CHG, and CHH sites and bisulfiteconversion rates were performed using the HT Analyzer BIQ (Lutsik et al,2011) software. Quality control of raw data and all statisticalanalysis.

They performed using the statistical language R and bioconductorsoftware, http:///www.bioconductor.org.

The selection of differentially methylated sites was based on thecalculation of Fisher's Exact test statistic Test, considering thosesites differentially methylated with a p-value set using Benjamini andHochberg method (1995) below 0.05. The □-value represented in Heatmapsgraphs is the ratio of methylated sequences with respect to the overallsum of methylated and unmethylated sequences per site (Du et al, 2010,BMC Bioinformatics, 30; 11: 587), i.e. β_(i,j)=M/(M OR) Where M is thenumber of methylated sequences at the site (i) and MID (j), and U is thenumber of non-methylated at the site (i) and MID (j) sequences.

Example 1: DNA Methylation is Increased in the D-Loop Region and Reducedin the ND1 Gene in Cases with Early Stages of AD-Related Pathology

An increase in methylation in CpG and non CpG sites (CHG and CHH) wasobserved in the D-loop region in cases with AD pathology related tostages I/II and III/IV of Braak (FIG. 1).

The degree of methylation was higher in cases of pathology related to ADwith respect to the control samples, and higher stage I/II versus stageIII/IV, as shown in log 2 graphics (OR) (FIG. 2). However, nodifferences in methylation of CHH sites between controls and cases withAD-related pathology at stages III/IV were found.

ND1 analysis revealed the presence of some less methylated CpG and CHGsites in cases with related AD pathology stage I/II and III/IV comparedto control samples (log 2 [O]>0, FIG. 3). No differences were found forthe CHH sites.

Example 2: DNA Methylation is Reduced in the D-Loop Region in theSubstantia Nigra of PD Cases

In contrast to what was observed in the entorhinal cortex in AD, theD-loop region showed a loss of methylation in almost all CpG and non CpGsites in the substantia nigra ub PD cases with respect to the controlsamples (FIG. 3). However, as with AD, the percentage of DNA methylationrepresents a small part of the total mitochondrial DNA.

Example 3: DNA Methylation is Increased in the Region D-Loop in MurineModels of AD

In this study, mice APP/PS1 (murine model of AD) six months of age (n=4)and control mice of the same age (n=4) were used. As can be seen in FIG.4A, the CpG sites showed hypermethylation in samples obtained frompathology of mouse models with respect to the control samples. Theseresults were also seen in CHG sites (see FIG. 4B).

To determine the methylation pattern as advance of AD, they employedmice APP/PS1 of three, six and twelve months. At three months, theAPP/APS1 mice have a low degree of accumulation of neuritic plaques,which increases in APP/APS1 mice six months old. AD model mice sixmonths of age show cognitive and memory failures. Finally, APP/APS1animals of 12 months show similar symptoms to those observed in humansin advanced stages of AD.

The analysis of the degree of methylation in the D-Loop regioncorroborated the pattern observed in samples of human brain tissue. Asshown in FIG. 5, a tendency to hypomethylation of CpG sites CHG and CHHwas observed in model mice in advanced stages of AD with respect to saidCpG, CHG and CHH sites model mice in early stages of AD (FIGS. 5A, 5Band 5C).

Example 4: The Presence of the Polymorphic Position 16519 inMitochondrial DNA is Associated with the Development of Alzheimer'sDisease

The results shown in Table 9 show that the presence of the polymorphismT16519C is associated with Alzheimer's disease. Said results wereobtained by conventional sequencing and using a chi-square test.

TABLE 9 Presence of polymorphism in samples obtained from individualscontrols and individuals with Alzheimer's disease (AD) at differentstages (Roman numerals). Type of sample presence of Number of Samplespolymorphism T16519C analyzed) (%) p-value Controls (n = 46) 56.5 — ADI/II (n = 47) 76.6 0.02 AD III/IV (n = 47) 74.4 0.03 AD V/VI (n = 46)76.1 0.02

The presence of heteroplasmy was observed in some cases and wasperformed again genotyping samples by using PCR-RFLP. The presence ofthe C allele creates a restriction site for the restriction enzymeHaeIII. The amplification of the sequence shown in SEQ ID NO: 30 wascarried out using oligonucleotides SEQ ID NO: 31 and SEQ ID NO: 32. Thefollowing band patterns depending on the genotype were obtained:

Genotype T: 183 bp, 318 bp

Genotype C: 61 bp, 183 bp, 257

Heteroplasmia: 61 bp, 183 bp, 257 bp, 319 bp.

What is claimed is:
 1. A method for detecting hypermethylation in theD-loop region of mitochondrial DNA comprising: (a) obtaining amitochondrial DNA sample comprising SEQ ID NO: 1 from a subject; and (b)detecting hypermethylation at CpG sites in the D-loop of themitochondrial DNA, wherein the CpG sites are selected from the groupconsisting of CpG sites at positions 16427, 16449, 16454, 16495, 16542,16565, 61, 162, and 170 of SEQ ID NO: 1; wherein said detecting isconducted by a technique selected from the group consisting ofmethylation specific PCR, bisulfite sequencing, techniques based onrestriction-digestion, pyrosequencing, assay ChIP-on-chip, differentialconversion, differential restriction and differential weight of site(s)methylated.
 2. A method of delaying progression of Alzheimer's diseasein a subject, comprising: (a) performing the method of detectinghypermethylation in the D-loop region of mitochondrial DNA according toclaim 1; and (b) delivering a treatment for delaying the progression ofAlzheimer's disease to the subject, wherein said treatment isadministration of a pharmaceutical agent selected from the groupconsisting of a cholinesterase inhibitor, a cholinesterase antagonistand N-methyl-D-aspartate (NMDA).
 3. The method of claim 2, wherein thetreatment comprises administration of a cholinesterase inhibitorselected from the group consisting of donepezil hydrochloride (Arecept),rivastigmine (Exelon) and galantemina (Reminyl).
 4. The method accordingto claim 2, wherein the subject has been diagnosed with Alzheimer'sdisease in stage I-II.
 5. The method according to claim 2, furthercomprising detecting hypomethylation at CpG sites in the ND1 gene ofmitochondrial DNA, wherein the CpG sites are selected from CpG sites atpositions 3351, 3375, 3379, 3406, 3453, 3549, and 3642 of SEQ ID NO: 1.6. The method according to claim 2, further comprising detecting (i)hypermethylation at CHG sites in the D-loop region of mitochondrial DNA,wherein the CHG sites are selected from CHG sites at positions of 16426,16453, 16459, 16466, 16479, 16514, 6, 33, 64, 104, 122, 128, 141, and253 of SEQ ID NO: 1, (ii) hypermethylation at CHH sites in the D-loopregion of mitochondrial DNA, wherein the CHH sites are selected from CHHsites at positions 16419, 16425, 16429, 16439, 16442, 16446, 16451,16458, 16465, 16478, 16498, 16507, 16511, 16520, 16527, 16536, 16540,16546, 16549, 16560, 16563, 4, 11, 15, 18, 26, 29, 39, 43, 48, 76, 86,110, 113, 132, 140, 144, 147, 150, 164, 167, 190, 194, and 198 of SEQ IDNO: 1, (iii) hypomethylation at CHG sites in the ND1 gene ofmitochondrial DNA, wherein the CHG sites are selected from CHG sites atpositions 3374, 3435, 3524, 3529, 3589, 3641, and 3657 of SEQ ID NO: 1,or (iv) hypomethylation at CpG sites in the ND1 gene of mitochondrialDNA, wherein the CpG sites are selected from CpG sites at positions3351, 3375, 3379, 3406, 3453, 3549, and 3642 of SEQ ID NO:
 1. 7. Themethod according to claim 1, wherein said detecting hypermethylation atCpG sites in the D-loop of the mitochondrial DNA, comprises detectinghypermethylation at positions 16427, 16449, 16454, 16495, 16542, 16565,61, 162, and 170 of SEQ ID NO:
 1. 8. The method according to claim 1,further comprising detecting hypermethylation at CpG sites in the ND1gene of the mitochondrial DNA, wherein the CpG sites are selected fromthe group consisting of CpG sites at positions 3351, 3375, 3379, 3406,3453, 3549, and 3642 of SEQ ID NO:
 1. 9. The method according to claim1, further comprising detecting hypermethylation at CHG sites in theD-loop region of the mitochondrial DNA, wherein the CHG sites areselected from the group consisting of CHG sites at positions 16426,16453, 16459, 16466, 16479, 16514, 6, 33, 64, 104, 122, 128, 141, and253 of SEQ ID NO:
 1. 10. The method according to claim 1, furthercomprising detecting hypermethylation at CHG sites in the ND1 gene ofthe mitochondrial DNA, wherein the CHG sites are selected from the groupconsisting of CHG sites at positions 3374, 3435, 3524, 3529, 3589, 3641,and 3657 of SEQ ID NO:
 1. 11. The method according to claim 1, furthercomprising detecting hypermethylation at CHH sites in the D-loop regionof the mitochondrial DNA, wherein the CHH sites are selected from thegroup consisting of CHH sites at positions 16419, 16425, 16429, 16439,16442, 16446, 16451, 16458, 16465, 16478, 16498, 16507, 16511, 16520,16527, 16536, 16540, 16546, 16549, 16560, 16563, 4, 11, 15, 18, 26, 29,39, 43, 48, 76, 86, 110, 113, 132, 140, 144, 147, 150, 164, 167, 190,194, 198 of SEQ ID NO:
 1. 12. The method of claim 1, wherein saiddetecting is conducted by pyrosequencing.
 13. The method according toclaim 1, wherein the mitochondrial DNA obtained from the subject iscontained in a biofluid or a biopsy of a solid tissue.
 14. The methodaccording to claim 13, wherein said biofluid is a peripheral blood or acerebrospinal fluid.
 15. The method according to claim 13, wherein saidsolid tissue is a brain tissue.
 16. The method according to claim 1,wherein step (b) comprises sequencing at least 11,672 reads per D-loopof the mitochondrial DNA.
 17. The method according to claim 1, furthercomprising detection of: (i) hypermethylation at CHG sites in the D-loopregion of mitochondrial DNA, wherein the CHG sites are selected from CHGsites at positions 16426, 16453, 16459, 16466, 16479, 16514, 6, 33, 64,104, 122, 128, 141, and 253 of SEQ ID NO: 1; (ii) hypermethylation atCHH sites in the D-loop region of mitochondrial DNA, wherein the CHHsites are selected from CHH sites at positions 16419, 16425, 16429,16439, 16442, 16446, 16451, 16458, 16465, 16478, 16498, 16507, 16511,16520, 16527, 16536, 16540, 16546, 16549, 16560, 16563, 4, 11, 15, 18,26, 29, 39, 43, 48, 76, 86, 110, 113, 132, 140, 144, 147, 150, 164, 167,190, 194, 198 of SEQ ID NO: 1; (iii) hypomethylation at CpG sites in theND1 gene of mitochondrial DNA, wherein the CpG sites are selected fromCpG sites at positions 3351, 3375, 3379, 3406, 3453, 3549, and 3642 ofSEQ ID NO: 1; (iv) hypomethylation at CHG sites in the ND1 gene ofmitochondrial DNA, wherein the CHG sites are selected from CHG sites atpositions 3374, 3435, 3524, 3529, 3589, 3641, and 3657 of SEQ ID NO: 1;(v) hypomethylation at CHG sites in the D-loop region of mitochondrialDNA, wherein the CHG sites are selected from CHG sites at positions16426, 16453, 16459, 16466, 16479, 16514, 6, 33, 64, 104, 122, 128, 141,and 253 of SEQ ID NO: 1; or (vi) hypomethylation at CHH sites in theD-loop region of mitochondrial DNA, wherein the CHH sites are selectedfrom CHH sites at positions 16419, 16425, 16429, 16439, 16442, 16446,16451, 16458, 16465, 16478, 16498, 16507, 16511, 16520, 16527, 16536,16540, 16546, 16549, 16560, 16563, 4, 11, 15, 18, 26, 29, 39, 43, 48,76, 86, 110, 113, 132, 140, 144, 147, 150, 164, 167, 190, 194, 198 ofSEQ ID NO:
 1. 18. A method of delaying progression of Parkinson'sdisease in a subject, comprising: (a) obtaining a mitochondrial DNAsample comprising SEQ ID NO: 1 from a subject; (b) detectinghypomethylation at CpG sites in the D-loop of the mitochondrial DNA,wherein the CpG sites are selected from the group consisting of CpGsites at positions 16427, 16449, 16454, 16495, 16542, 16565, 61, 162,and 170 of SEQ ID NO: 1; and (c) delivering a treatment for delaying theprogression of Parkinson's disease to the subject, wherein saidtreatment is administration of a pharmaceutical agent selected from thegroup consisting of L-dopa, an inhibitor of catechol-o-methyltransferase (COMT), an inhibitor of monoamine oxidase B (MAOB) and adopamine agonist, wherein said detecting is conducted by a techniqueselected from the group consisting of methylation specific PCR,bisulfite sequencing, techniques based on restriction-digestion,pyrosequencing, assay ChIP-on-chip, differential conversion,differential restriction and differential weight of site(s) methylated.19. The method according to claim 18, wherein the subject has beendiagnosed with Parkinson's disease in stage III-V.
 20. The method ofclaim 18, wherein the treatment comprises administration of an inhibitorof catechol-o-methyl transferase (COMT) selected from the groupconsisting of tolcapone (Tasmar) and entacapone (Comtan).
 21. The methodof claim 18, wherein the treatment comprises administration of aninhibitor of monoamine oxidase B (MAOB) selected from the groupconsisting of selegiline (Eldepryl) and rasagaline (Azilect).
 22. Themethod of claim 18, wherein the treatment comprises administration of adopamine agonist selected from the group consisting of pramipexole,rotigotine and ropinirole.
 23. The method of claim 18, furthercomprising repeating steps (a) and (b) at a later stage of Parkinson'sdisease.
 24. The method according to claim 18, further comprisingdetection of (i) hypomethylation at CHG sites in the D-loop ofmitochondrial DNA, wherein the CHG sites are selected from CHG sites atpositions 16426, 16453, 16459, 16466, 16479, 16514, 6, 33, 64, 104, 122,128, 141, and 253 of SEQ ID NO: 1; and/or (ii) hypomethylation at CHHsites in the D-loop of mitochondrial DNA, wherein the CHH sites areselected from CHH sites at positions 16419, 16425, 16429, 16439, 16442,16446, 16451, 16458, 16465, 16478, 16498, 16507, 16511, 16520, 16527,16536, 16540, 16546, 16549, 16560, 16563, 4, 11, 15, 18, 26, 29, 39, 43,48, 76, 86, 110, 113, 132, 140, 144, 147, 150, 164, 167, 190, 194, 198of SEQ ID NO: 1.